-
Accepted Manuscript
Cholesterol-lowering effects of a putative probiotic strain
Lactobacillus plantarum EMisolated from kimchi
Eun A. Choi, Hae Choon Chang
PII: S0023-6438(15)00035-3
DOI: 10.1016/j.lwt.2015.01.019
Reference: YFSTL 4400
To appear in: LWT - Food Science and Technology
Received Date: 30 June 2014
Revised Date: 2 January 2015
Accepted Date: 13 January 2015
Please cite this article as: Choi, E.A, Chang, H.C.,
Cholesterol-lowering effects of a putative probioticstrain
Lactobacillus plantarum EM isolated from kimchi, LWT - Food Science
and Technology (2015),doi: 10.1016/j.lwt.2015.01.019.
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http://dx.doi.org/10.1016/j.lwt.2015.01.019
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1
Cholesterol-lowering effects of a putative probiotic strain
2
Lactobacillus plantarum EM isolated from kimchi 3
4
5
6
Eun A Choi Hae Choon Chang* 7
8
9
10
Department of Food and Nutrition, Kimchi Research Center, Chosun
University, 309 11
Pilmun-daero, Dong-gu, Gwangju 501-759, South Korea 12
13
* Corresponding author: Tel.: +82 62 2307345; fax: +82 62
2228086 14
e-mail: [email protected]
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Abstract 16
17
Lactobacillus plantarum EM, which was isolated from kimchi,
showed high cholesterol removal by 18
growing, resting, and even dead cells. Moreover, cell wall
fraction of L. plantarum EM removed 19
cholesterol in a cell wall concentration-dependent pattern.
Lactobacillus acidophilus ATCC 43121 as a 20
control showed high cholesterol removal by growing cells,
whereas resting and dead cells showed less 21
cholesterol removal. Scanning electron micrographs showed that
large amounts of cholesterol adhered to 22
surfaces of growing and dead cells of L. plantarum EM without
changes in cell morphology. On the other 23
hand, only a small amount of cholesterol adhered to the surface
of L. acidophilus ATCC 43121 cells, and 24
growing cells of L. acidophilus ATCC 43121 showed morphological
changes with a thinner and longer 25
shape. Based on these results, cholesterol removal mechanisms by
L. plantarum EM could be attributed to 26
enzymatic assimilation including bile salt hydrolase activity
and cell surface-binding. Further, L. 27
plantarum EM showed reasonable tolerance to acid and bile
stresses and displayed antagonistic activity 28
against pathogens. Moreover, L. plantarum EM did not represent a
health risk due to antibiotic resistance. 29
The results of this study indicate that L. plantarum EM may be a
promising probiotic candidate and 30
adjunct culture for reduction of serum cholesterol level
regardless of its viability. 31
32
33
34
Keywords: Cholesterol-lowering effect, Lactobacillus plantarum,
Probiotic, Kimchi35
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1. Introduction 36
37
Lactic acid bacteria (LAB) are industrially important
microorganisms worldwide for the fermentation 38
of foods. Moreover, LAB as probiotics have become the focus of
intensive international research for their 39
health-promotion effects, which include reduction of serum
cholesterol level, stimulation of immune 40
responses, cancer prevention, and alleviation of diarrhea (De
Vrese & Schrezenmeir, 2008). The 41
numerous health benefits of LAB have made them into promising
probiotic candidates that are studied for 42
their desirable properties. The World Health Organization (WHO)
has predicted that cardiovascular 43
disease will remain the leading cause of death through 2030,
affecting approximately 23.6 million people 44
worldwide (WHO, 2009). Increased serum cholesterol correlates
highly with coronary heart disease 45
(Kumar et al., 2012). Thus, investigators are paying close
attention to the cholesterol-lowering effects of 46
LAB among their many functional effects. 47
Studies have indicated that several LAB strains, mainly
Lactobacillus spp., have cholesterol-reducing 48
effects in vitro or in vivo (Kumar et al., 2012; Ooi &
Liong, 2010). However, the exact mechanisms of 49
serum cholesterol reduction by probiotic bacteria are not
completely clear. Several mechanisms have been 50
proposed in vitro, including assimilation (Pereira & Gibson,
2002), surface binding (Liong & Shah, 2005), 51
incorporation into cellular membranes (Lye, Rusul, & Liong,
2010a), co-precipitation with deconjugated 52
bile (Liong & Shah, 2006), enzymatic deconjugation of bile
acids by bile salt hydrolase (BSH) (Lambert, 53
Bongers, de Vos, & Kleerebezem, 2008), conversion of
cholesterol into coprostanol (Lye, Rusul, & Liong, 54
2010b), and production of short-chain fatty acids by probiotics
(De Preter et al., 2007). 55
The aim of this study was to investigate high cholesterol
removal by dead cells of the selected LAB 56
strain as well as its possible mechanisms of action. Moreover,
the selected strain was identified and 57
evaluated in terms of its probiotic properties, such as acid and
bile tolerance, antimicrobial activity 58
against pathogens, and antibiotic resistance. 59
60
2. Materials and methods 61
62
2.1. Bacterial cultures and screening of LAB strains for
cholesterol removal 63
64
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Screening of LAB strains from kimchi for cholesterol removal was
carried out by determination of 65
BSH activity (Dashkevicz & Feighner, 1989) and cholesterol
assimilation (Rudel & Morris, 1973). 66
67
2.1.1. BSH activity 68
69
For BSH activity measurement, 10 l of culture grown in MRS broth
was spotted onto BSH screening 70
medium, which consisted of de Man Rogosa and Sharpe (MRS; Difco,
Sparks, MD, USA) agar 71
supplemented with 0.5% (w/v) sodium salt taurodeoxycholic acid
(TDCA, Sigma-Aldrich, St Louis, MO, 72
USA) and 0.37 g/l of CaCl2 (Dashkevicz & Feighner, 1989).
Plates were incubated anaerobically at 37 C 73
using GasPak EZ anaerobe container systems (Becton, Dickinson
and Company, Sparks, MD, USA), 74
after which BSH activity was determined by measuring the
diameters of precipitation zones. BSH activity 75
was expressed based on the diameters of precipitation zones on
BSH screening medium: -, no 76
precipitation; +, precipitation zone of up to 10 mm; ++,
precipitation zone of up to 15 mm; and +++, 77
precipitation zone of up to 20 mm. 78
79
2.1.2. Cholesterol assimilation 80
81
Cholesterol assimilation was determined using the method of
Rudel and Morris (Rudel & Morris, 82
1973). LAB cells grown overnight were inoculated (1%) and
cultivated anaerobically using GasPak EZ 83
anaerobe container systems (Becton, Dickinson and Company) at 37
C in MRS broth supplemented with 84
0.5% (w/v) oxgall (Sigma-Aldrich) and 0.1 g/l of water-soluble
cholesterol (Sigma-Aldrich). Following 85
incubation, cells were harvested (9,950 g, 5 min, 4 C), after
which 1 ml of supernatant was added to 86
2 ml of 33% (w/v) potassium hydroxide and 3 ml of 95% (v/v)
ethanol. The mixture was shaken well for 87
1 min and then heated for 10 min in a 60 C water bath. After
cooling in cold water, 5 ml of hexane was 88
added, followed by mixing and addition of 1 ml of distilled
water. The tube was allowed to stand for 10 89
min at room temperature for phase separation, after which 3 ml
of the hexane phase was transferred to a 90
clean tube. The hexane phase was then evaporated under a
nitrogen stream. The concentrated phase was 91
added to 4 ml of freshly prepared O-phthalaldehyde
(Sigma-Aldrich, 0.5 mg of O-phthalaldehyde/ml of 92
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acetic acid), mixed, and permitted to stand at room temperature
for 10 min. Following the addition of 93
2 ml of concentrated sulfuric acid and incubation for 10 min,
the absorbance at 550 nm was read using a 94
spectrophotometer (Amersham Biosciences, Uppsala, Sweden).
Absorbance values were compared to 95
those obtained using cholesterol standard. 96
97
2.2. Identification of isolate 98
99
The LAB strain showing the highest BSH activity and cholesterol
assimilation during measurement of 100
cholesterol removal was identified for further study.
Identification was carried out by Gram staining, 101
catalase test, and morphological observation under a microscope,
and 16S rRNA gene sequences were 102
determined using an API prism 3730 DNA analyzer (Applied
Biosystems, Foster City, CA, USA) 103
according to the method described by Yang and Chang (Yang &
Chang, 2008). The determined 16S rRNA 104
gene sequences were compared with sequences available in the
GenBank database using the Clustal W 105
program (Thompson, Higgins, & Gibson, 1994). 106
107
2.3. Growth in bile acids and cholesterol 108
109
Growth of LAB under bile acids and cholesterol was determined.
LAB grown overnight in MRS were 110
inoculated (1%) into MRS broth supplemented with 0.3% (w/v)
oxgall and 0.1 g/l of water-soluble 111
cholesterol, 0.5% (w/v) oxgall and 0.1 g/l of water-soluble
cholesterol, 0.3% (w/v) TDCA and 0.1 112
g/l of water-soluble cholesterol, and 0.5% (w/v) TDCA and 0.1
g/l of water-soluble cholesterol. 113
Growth of LAB in MRS broth without bile was used as a control.
Initial pHs of the prepared media were 114
pH 6.2-6.4. The culture was incubated anaerobically for 24 h at
37 C, after which the absorbance at 600 115
nm was measured. 116
117
2.4. Acid and bile tolerance 118
119
Tolerance levels of LAB to acid and bile salts were determined
as previously described (Ryu & Chang, 120
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2013). LAB were cultivated at 37 C for 24 h in MRS broth and
harvested by centrifugation (9,950 g, 5 121
min, 4 C), after which cell pellets were resuspended in
phosphate-buffered saline (PBS, pH 2.5; Hyclone, 122
Logan, UT, USA), simulated gastric juice (SGJ; 3 mg of pepsin
dissolved in 1 ml of 0.5% saline buffer, 123
pH 2.5), or bile salt (0.3% oxgall dissolved in PBS, pH 8.0)
with approximately 9.8 log CFU/ml of cell 124
numbers. The suspensions were incubated at 37 C for 1 h in PBS
or SGJ for 3 h in bile salt. Thereafter, 125
suspensions were harvested (9,950 g, 5 min, 4 C) and resuspended
in MRS broth, after which viable 126
cell counts were enumerated on MRS agar after incubation at 37 C
for 48 h. LAB suspended in MRS 127
broth without acid or bile salt were considered as controls.
128
129
2.5. Antibiotic susceptibility 130
131
Antibiotic susceptibility of the selected LAB isolate was
determined according to the technical 132
guidelines of the European Food Safety Authority (EFSA) (EFSA,
2012) using a previously described 133
method (Ryu & Chang, 2013). The minimal inhibitory
concentrations (MIC) of antibiotics were 134
measured. After culturing LAB in MRS broth for 24 h, cells were
centrifuged and resuspended in Muller 135
Hinton (MH; Difco) broth containing 0.5% dextrose. Resultant
cell suspensions were diluted in the same 136
medium to a cell density of 5.0 log CFU/ml (A600=0.4-0.5). Each
antibiotic was added to the diluted cell 137
suspension, which was incubated anaerobically at 37 C for 24-48
h. Cell growth was observed visually 138
and measured based on absorbance at 600 nm (Amersham
Biosciences). MIC values were defined as the 139
lowest antibiotic concentrations where no bacterial growth
occurred. All antibiotics were purchased from 140
Sigma-Aldrich. 141
142
2.6. Antimicrobial activity 143
144
LAB grown overnight were inoculated (1%) into MRS broth and
incubated anaerobically at 37 C for 145
24 h. The culture was centrifuged, after which the supernatant
was membrane-filtered (0.4 m membrane 146
filter; Advantec MFS, Dublin, CA, USA) and then freeze-dried
(Samwon, Busan, South Korea). The 147
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freeze-dried culture supernatant was concentrated (5-fold) in 20
mM sodium acetate (pH 4.0). 148
Concentrated (5-fold) MRS broth was used as a control. 149
Antimicrobial activities against seven pathogens were
investigated using spot-on-the-lawn assay 150
(Hoover & Harlander, 1993). Pathogens were grown at 37 C on
Luria-Bertani (LB; Difco) agar for 151
E.coli, Pseudomonas aeruginosa, and Salmonella enterica serovar
Typhi, nutrient agar (NA; Difco) 152
supplemented with 2% (w/v) NaCl for Vibrio parahaemolyticus, and
tryptic soy agar (TSA; Difco) for 153
Bacillus cereus, Micrococcus luteus, and Staphylococcus aureus.
ATCC strains were purchased from the 154
American Type Culture Collection (Manassas, VA, USA), whereas
KCTC strains were purchased from 155
the Korean Collection for Type Cultures (Daejeon, South Korea).
LB, TSA, or NA plates were spread 156
with each pathogen at a concentration of 6 log CFU/ml. An
aliquot (10 l) of LAB culture was spotted 157
onto sensitive pathogen plates for each antimicrobial sample.
Antimicrobial activity, expressed as 158
arbitrary units (AU) per milliliter, was defined as the
reciprocal of the highest dilution at which pathogen 159
growth was inhibited (Ammor, Tauveron, Dufour, & Cherallier,
2006). The antimicrobial titer was 160
calculated as (1000/d) D, where D is the dilution factor and d
is the dose (amount of antimicrobial 161
samples pipetted onto each spot). The above experiment was
performed in triplicate. 162
163
2.7. Cholesterol removal by growing, resting, and dead cells
164
165
For cholesterol assimilation by growing cells, LAB cells were
inoculated (1%) and grown 166
anaerobically at 37 C in MRS broth supplemented with 0.5% (w/v)
oxgall and 0.1 g/l of water-soluble 167
cholesterol as well as in MRS broth containing 0.5% (w/v) TDCA
and 0.1 g/l of water-soluble cholesterol. 168
To prepare resting and dead cells (Lye, Rusul, & Liong,
2010a), LAB strains were incubated 169
anaerobically at 37 C in MRS broth containing 0.5% oxgall as
well as in MRS broth containing 0.5% 170
TDCA. The cell pellet obtained from the media was washed twice
with sterile-distilled water. For 171
cholesterol removal by resting cells, the cell pellet was
suspended in 0.05 M phosphate buffer (pH 6.8) 172
containing 0.5% oxgall and 0.1 g/l of water-soluble cholesterol
as well as in 0.5% TDCA and 0.1 g/l of 173
water-soluble cholesterol. For dead cell assay, the cell pellet
was suspended in saline and heat-killed at 174
121 C for 15 min. The dead cells were harvested, after which the
pellet was suspended in MRS 175
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containing 0.5% oxgall and 0.1 g/l of water-soluble cholesterol
as well as in 0.5% TDCA and 0.1 g/l 176
water-soluble cholesterol. 177
All strains were incubated at 37 C for 24 h, after which the
mixture was centrifuged (9,950 g, 5 min, 178
4 C). The remaining cholesterol concentration of broth was
determined using the method of Rudel and 179
Morris (Rudel & Morris, 1973) described in 2.1.1.
Cholesterol assimilation (Ooi & Liong, 2010) was 180
expressed as {0.1 g/l of cholesterol (control) concentration of
remaining cholesterol in cultures}/control 181
100. 182
183
2.8. Cholesterol removal by LAB cell wall 184
185
2.8.1. Preparation of cell wall 186
187
Isolation of LAB cell wall was carried out as previously
described with modifications (Piuri, Sanchez-188
Rivas, & Ruzal, 2005). Exponential phase of LAB cultures (20
ml) was harvested, washed twice with 189
PBS (pH 7.0-7.2, Hyclone), and suspended in 20 ml of PBS at 4 C.
Ice-jacketed cell suspensions were 190
sonicated (Amp 60, 10 min, pulse 2 sec; Sonics, Newton, CT, USA)
twice, centrifuged, suspended in 5% 191
(w/v) sodium dodecyl sulfate (SDS, Roche, Indianapolis, IN,
USA), and then incubated at 60 C for 30 192
min. The crude cell wall fraction was centrifuged (9,950 g, 30
min, 4 C), washed twice with PBS, 193
suspended in 10 mM Tris-HCl (pH 7.5) with 10 mM MgCl2, and
subsequently incubated with a mixture 194
of 0.01 g/ml of DNase (Sigma-Aldrich), 0.01 mg/ml of RNase A
(Sigma-Aldrich), and 0.01 g/ml of 195
trypsin (Sigma-Aldrich) for 4 h at 37 C. Finally, the cell wall
fraction was centrifuged, washed three 196
times with PBS, suspended in 20 ml of PBS, and then homogenized
twice by sonication (Amp 60, 10 min, 197
pulse 2 sec). The purified cell wall fraction was then
freeze-dried. 198
199
2.8.2. Cholesterol removal by LAB cell wall fraction 200
201
The prepared cell wall fraction was suspended in MRS containing
0.5% TDCA and 0.1 g/l of water-202
soluble cholesterol along with A600=1, 2, 3, 4, and 5 mg/ml of
the LAB cell wall fraction. The suspension 203
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was incubated anaerobically at 37 C for 24 h, after which the
mixture was centrifuged (9,950 g, 30 min, 204
4 C). The remaining cholesterol concentration of the supernatant
was measured using the method of 205
Rudel and Morris (Rudel & Morris, 1973), and its cholesterol
assimilation (%) was determined. 206
207
2.9. Scanning electron microscopy (SEM) 208
Growing and dead cells as well as cell wall fraction were
prepared as described in sections 2.7 and 2.8. 209
The prepared growing and dead cells (A6008.0) as well as cell
wall fraction (A60012.0) were suspended 210
in MRS broth supplemented with 0.5% TDCA and 0.1 g/l of
cholesterol incubated at 37 C for 24 h. The 211
prepared LAB cultures in MRS broth without any supplementation
were used as a control. After 212
incubation, LAB cultures were centrifuged and then washed twice
with PBS (pH 7.0-7.2, Hyclone). 213
Cholesterol attached to LAB cells or cell wall fraction was
observed using field emission scanning 214
electron microscopy (FE-SEM; Hitachi, Tokyo, Japan) as described
previously with slight modifications 215
(Kalchayanand, Dunne, Sikes, & Ray, 2004). LAB cell or cell
wall fraction was prefixed with 0.1 M 216
sodium cacodylate buffer (pH 7.4) containing 2.5% (v/v)
glutaldehyde (pH 7.4; Sigma-Aldrich) for 2 h at 217
4 C, followed by washing three times with 0.1 M sodium
cacodylate buffer (pH 7.4). The samples were 218
then postfixed with 1% (w/v) osmium tetroxide in 0.1 M sodium
cacodylate buffer (pH 7.4) for 1-2 h, 219
washed three times with 0.1 M sodium cacodylate buffer (pH 7.4),
and dehydrated in an ascending 220
ethanol series [(%, v/v): 50, 60, 70, 80, 90, 95, and 100
ethanol] for 2 min each. Finally, the samples were 221
immersed two times in 100% tert-butanol for 20 min and then
coated with platinum using a sputter coater 222
(E-1030 Ion sputter, Hitachi, Tokyo, Japan). The resultant chips
were viewed in a FE-SE microscope. 223
224
2.10. Statistical analysis 225
226
Data are presented as the means and standard deviations (means
SD) of three independent 227
experiments performed in duplicate or triplicate. Statistical
analysis was performed on the data using 228
Duncans Multiple Range Test (DMRT) by SPSS ver. 21.0 for Windows
(SPSS, Chicago, IL, USA) with 229
statistical significance determined at p < 0.05. 230
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231
3. Results and discussion 232
233
3.1. Isolation and identification of cholesterol-lowering LAB
234
235
Seventy LAB cultures were isolated from 28 kimchi samples
collected from 17 cities in South Korea. 236
All isolates were grown well at 30 C in MRS medium. However,
only 50 LAB strains among the 70 total 237
LAB strains could be grown at 37 C. These 50 LAB strains were
preselected for further experimentation. 238
BSH activity and cholesterol assimilation of the 50 preselected
LAB strains were examined (Table 1). Of 239
the 50 tested LAB strains, 16 LAB strains expressed BSH
activity, and their cholesterol assimilation 240
varied from 3.51-80.86%. Based on this result, we selected two
strains (EM and DC1) for further 241
experimentation. Among the 50 strains, EM showed the strongest
cholesterol assimilation (80.86%) and 242
BSH activity (17.2 mm precipitation zone), whereas DC1 showed
the weakest cholesterol assimilation 243
(3.51%) and BSH activity (5.3 mm precipitation zone). 244
The EM and DC1 strains were Gram-positive, rod-shaped, and
catalase-negative. When 16S rRNA 245
gene sequences of the EM (1,881 bp) and DC1 (1,471 bp) isolates
were determined and compared with 246
those of type strains in GenBank, EM and DC1 sequences showed
99.9% and 99.9% homologies with 247
those of L. plantarum JCM 1149T and L. sakei DSM 20017T,
respectively. Thus, EM and DC1 isolates 248
were finally designated as L. plantarum EM and Lactobacillus
sakei DC1, respectively, and their gene 249
sequences were deposited in GenBank (accession numbers KC 422389
for EM and KJ 812206 for DC1). 250
251
3.2. Acid and bile tolerance 252
253
It is well known that probiotic bacteria should be capable of
surviving passage through the 254
gastrointestinal tract based on acid tolerance to human gastric
juice as well as bile tolerance for survival 255
in the small intestine (Saarela, Mogensen, Fondn, Mtt, &
Mattila-Sandholm, 2000). Therefore, these 256
characteristics may serve as suitable criteria for probiotic
bacteria selection. L. plantarum EM showed 257
high tolerance to acidic conditions at pH 2.5 (PBS and SGJ)
after 1 h of exposure at 37 C (Table 2). 258
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When L. plantarum EM was added to PBS containing 0.3% oxgall (pH
8.0) for 3 h at 37 C, initial cell 259
counts (9.78 log CFU/ml) of L. plantarum EM decreased to 5.53
log CFU/ml. In this study, acid and bile 260
treatments to L. plantarum EM were carried out under nutritional
deficit. Considering the human 261
gastrointestinal tract is full of food, the actual survival of
L. plantarum EM in the gastrointestinal tract 262
will actually be greater than the result shown in Table 2. This
suggests that L. plantarum EM has 263
reasonable tolerance to acid and bile and should be active in
the gastrointestinal tract. 264
265
3.3. Antibiotic resistance 266
267
L. plantarum EM was assessed for its resistance to nine
antibiotics, including those highlighted by the 268
EFSA (Florez et al., 2006). As shown in Table 3, L. plantarum EM
was susceptible to all of the antibiotics 269
tested, except vancomycin. The technical guidelines of the EFSA
have demonstrated that no breakpoints 270
for vancomycin and streptomycin are required for L. plantarum
(EFSA, 2012). Therefore, it seems 271
reasonable to conclude that consumption of L. plantarum EM in
this study does not represent a health risk 272
to humans due to antibiotic resistance. It is recognized that
probiotics lack undesirable properties such as 273
antibiotic resistance (Saarela, Mogensen, Fondn, Mtt, &
Mattila-Sandholm, 2000; Florez et al., 2006). 274
Thus, any evaluation of new probiotic candidates, including
those traditionally used in food fermentation, 275
should carefully confirm their safety status. 276
277
3.4. Antimicrobial activity 278
279
L. plantarum EM showed strong activities against the seven
tested pathogens (Table 4). In particular, L. 280
plantarum EM showed its strongest activity against Vibrio
parahaemolyticus ATCC 17802 (25,600 units) 281
as well as its weakest activity against Staphylococcus aureus
ATCC 29123 (200 units). L. plantarum EM 282
showed a broad antimicrobial spectrum against Gram-positive and
Gram-negative pathogens (Table 4). 283
Antimicrobial substances produced from LAB are known to include
H2O2, CO2, lactic and acetic acids, 284
bacteriocin, and others (Usman & Hosono, 1999). However,
most of these antimicrobial substances from 285
LAB strains have a narrow antimicrobial spectrum, and thus their
inhibitory effects are only against 286
closely related species (Holzapfel, Geisen, & Schillinger,
1995). One of the most important functional 287
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requirements of probiotics is a broad antimicrobial spectrum as
well as antagonism against pathogenic 288
bacteria with strong antimicrobial activity (Saarela, Mogensen,
Fondn, Mtt, & Mattila-Sandholm, 289
2000). Thus, the results represented in Table 4 for L. plantarum
EM fulfill the beneficial requirements of 290
probiotics. 291
292
3.5. Growth in the presence of bile acid 293
294
Growth in the presence of bile acid (oxgall or TDCA) was
examined using L. plantarum EM and L. 295
sakei DC1 (Table 5). L. acidophilus ATCC 43121, which is
characterized by high cholesterol assimilation 296
(Gilliand & Walker, 1989; Gilliand, Nelson, & Maxwell,
1985), was used as a control strain. Generally, 297
all three strains grew well in the presence of both bile acids.
However, growth of the three LAB strains 298
slightly decreased in TDCA- or oxgall-containing media. Oxgall
showed a slightly higher toxic effect on 299
LAB growth than TDCA. The antimicrobial action of bile is well
known; bile salt at high concentrations 300
can rapidly dissolve membrane lipids and cause dissociation of
integral membrane proteins, resulting in 301
leakage of cell contents and cell death (Begley, Gahan, &
Hill, 2005). The results represented in Table 5 302
suggest that the three LAB strains are moderately resistant to
bile toxicity. 303
304
3.6. Cholesterol removal 305
306
3.6.1. Cholesterol removal by growing, resting, and dead cells
307
308
As shown in Table 6, cholesterol removal according to cell state
was in the order of growing, resting, 309
and dead cells. L. plantarum EM showed the highest cholesterol
removal among the three tested strains 310
regardless of cell state. As already shown in Table 5, L. sakei
DC1 showed approximately 1/2 growth 311
(A6005) compared to the other two LAB strains (A6008-10).
However, cholesterol removal of L. sakei 312
DC1 was much lower (1/3-1/30) than that of L. plantarum EM or L.
acidophilus ATCC 43121 (Table 6). 313
Cholesterol removal by growing cells of L. acidophilus ATCC
43121 was as high as that of L. plantarum 314
EM. However, cholesterol removal by dead cells of L. acidophilus
ATCC 43121 was significantly (p < 315
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0.05) lower than that of L. plantarum EM. 316
317
3.6.2. Cholesterol removal by LAB cell wall fraction and SEM
observation 318
319
To determine the reason for such high cholesterol removal by
dead cells of L. plantarum EM, 320
cholesterol removal by cell wall fractions of both L. plantarum
EM and L. acidophilus ATCC 43121 was 321
investigated. As shown in Fig. 1, the cell wall fraction of L.
plantarum EM removed cholesterol in a cell 322
wall concentration-dependent pattern. On the other hand, the
cell wall fraction of L. acidophilus ATCC 323
43121 showed significantly (p < 0.05) lower cholesterol
removal than that of L. plantarum EM. This 324
result indicates that cholesterol can be removed by the cell
wall fraction itself without any metabolic 325
process. 326
Thus, we tried to observe cholesterol attachment to LAB cells
according to cell state (growing, dead 327
cell, and cell wall fraction) using SEM. As shown in Fig. 2,
large amounts of cholesterol adhered to the 328
surfaces of growing and dead cells of L. plantarum EM. Adherence
of cholesterol to the cell wall fraction 329
was observed only with L. plantarum EM, whereas no cholesterol
adherence or alteration of cell 330
morphology could be observed with L. sakei DC1. On the other
hand, only small amounts of cholesterol 331
adhered to surfaces of growing and dead cells of L. acidophilus
ATCC 43121. Interestingly, L. 332
acidophilus ATCC 43121 cells underwent morphological changes in
a growing state (Fig. 2 - growing cell 333
B), as evidenced by a longer and thinner cell shape (arrow
indicated) compared to control growing cells 334
of L. acidophilus ATCC 43121 (Fig. 2 - growing cell A). Further,
dead cells of L. acidophilus ATCC 335
43121 did not show any morphological changes. Noh et al.
previously reported the incorporation of 336
cholesterol into the cellular membrane of L. acidophilus ATCC
43121 in the presence of oxgall and 337
cholesterol (Noh, Kim, & Gilliand, 1997). Namely, a portion
of the assimilated cholesterol was recovered 338
in the membrane fractions of cells grown in media containing
cholesterol, which suggests that cholesterol 339
may have altered the cellular membrane or wall of L. acidophilus
ATCC 43121. 340
Cholesterol removal (%) by growing cells was almost the same
between L. plantarum EM and L. 341
acidophilus ATCC 43121 (Table 6). However, examination of
cholesterol adherence onto these two LAB 342
using SEM showed that their configurations were quite different.
These data indicate that the cholesterol 343
removal mechanism of L. plantarum EM is different from that of
L. acidophilus ATCC 43121. As 344
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reported already by Noh et al. (Noh, Kim, & Gilliand, 1997),
incorporation of cholesterol into the cellular 345
membrane of L. acidophilus ATCC 43121 during growth might result
in morphological changes, as 346
represented in this study (Fig. 2). On the other hand, Hosono
and Tano-oka reported cholesterol binding 347
to LAB varies among strains and species and hypothesized that
these differences in binding abilities can 348
be attributed to chemical and structural properties of cell wall
peptidoglycans containing amino acids 349
capable of binding to cholesterol (Hosono & Tano-oka, 1995).
Thus, strong cholesterol attachment to L. 350
plantarum EM regardless of its cell state might be due to the
unique in chemical and structural properties 351
of the L. plantarum EM cell wall compared to those of other LAB
cell types. However, direct 352
investigation of the chemical and structural properties of the
L. plantarum EM cell wall that promote 353
binding to cholesterol is required. Cholesterol removal by dead
cells of LAB strains has been reported 354
previously, even though the degree of removal is significantly
less compared to that of growing cells 355
(Zeng, Pan, & Guo, 2010; Liong & Shah, 2005; Mahdieh,
Hamid, & Naheed, 2014; Sirilun, Chaiyasut, 356
Kantachote, & Luxananil, 2010; Emami & Bazargani, 2014).
Further, cholesterol removal by dead cells 357
due to cholesterol attachment to the cellular membrane has been
investigated based on scanning electron 358
microscopic observation of cholesterol binding to growing cells
(Lye, Rusul, & Liong, 2010a; Ooi & 359
Liong, 2010). To the best of our knowledge, there is no direct
evidence verifying cholesterol removal by 360
dead cells via binding of cholesterol to dead cells or the cell
wall fraction itself. 361
Based on the results in Table 6 and Fig. 2, the
cholesterol-lowering effect of L. plantarum EM can be 362
considered to be a major part of enzymatic assimilation,
including BSH activity. Further, L. plantarum 363
EM showed a cholesterol-lowering effect with strong binding
capability to cell surfaces regardless of its 364
viability. Cholesterol bound to LAB strains has been shown to
inhibit intestinal absorption of cholesterol 365
in previous investigations (Usman & Hosono, 1999). Thus, the
high cholesterol-binding capability of L. 366
plantarum EM might be more effective in reducing the serum
cholesterol level in humans. However, in 367
vivo evaluation of this strain in humans is required to assess
the usage of probiotics for reducing serum 368
cholesterol. 369
370
4. Conclusions 371
372
As the term probiotics means for life, viable probiotics are
required to have beneficial effects on their 373
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host. In this study, L. plantarum EM showed high cholesterol
removal by growing, resting, and even dead 374
cells based on the high cholesterol-binding capacity of its cell
wall fraction. Therefore, L. plantarum EM 375
could be a potent probiotic to reduce serum cholesterol
regardless of its viability. Moreover, L. plantarum 376
EM also appeared to meet the functional criteria required for
beneficial probiotics, such as acid and bile 377
tolerance, antagonistic activity against pathogens, and
antibiotic susceptibility. 378
379
380
381
382
383
384
Acknowledgment 385
386
This research was supported by the Technology Development
Program for Food, Ministry of 387
Agriculture, Food and Rural Affairs, South Korea. 388
389
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Table 1 476 Cholesterol assimilation and BSH activities of LAB
strains isolated from kimchi. 477
*BSH activity was expressed based on the diameters of
precipitation zones on BSH screening medium: -, 478 no
precipitation; +, precipitation zone of up to 10 mm; ++,
precipitation zone of up to 15 mm; and +++, 479 precipitation zone
of up to 20 mm. 480
No. Strain Shape Cholesterol
Assimilation (%) BSH activity
1 AB rod 11.71 +
2 BC coccus 5.95 -
3 BY rod 8.33 -
4 C12 rod 10.54 -
5 C14 rod 15.50 -
6 CC rod 8.33 -
7 CM1 short rod 8.92 -
8 CM3 short rod 33.78 -
9 CS short rod 11.94 +
10 DC1 rod 3.51 +
11 DC2 rod 5.23 -
12 DC3 short rod 6.98 -
13 DJ1 coccus 13.20 -
14 DM1 coccus 5.14 -
15 DS rod 10.72 +
16 EM rod 80.86 +++
17 GJ2 coccus 53.83 +
18 GJ6 short rod 13.29 +
19 GJ7 rod 17.70 +
20 GS rod 7.07 -
21 HA1 short rod 8.78 -
22 HC coccus 10.54 -
23 HD1 rod 77.61 +++
24 HJ short rod 10.72 -
25 JD short rod 10.09 ++
26 JH short rod 8.92 -
27 JS coccus 9.41 -
28 JT coccus 10.81 -
29 JW short rod 10.86 +
30 KO short rod 7.39 -
31 KW coccus 10.45 -
32 MA short rod 9.73 -
33 MH short rod 9.59 -
34 MO1 short rod 17.25 -
35 MP1 coccus 6.80 -
36 MS1 short rod 9.14 -
37 NG1 coccus 15.32 -
38 NJ1 short rod 4.91 -
39 NJ6 short rod 13.15 -
40 NM1 short rod 15.86 ++
41 NO1 rod 78.11 ++
42 NP coccus 13.87 +
43 OS rod 16.49 -
44 PH2 rod 11.98 +
45 R1 coccus 9.37 -
46 SC coccus 3.83 -
47 SH rod 9.68 -
48 SM rod 11.08 +
49 UC short rod 3.78 -
50 W1 coccus 8.29 -
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Table 2 481 Acid and bile tolerance of L. plantarum EM. 482
Strain
Initial mean counts
Survival after 1 h at pH 2.5
Survival after 3 h at pH 8.0
PBSb
SGJc
0.3% oxgall
log CFU/ml
log CFU/ml %a
log CFU/ml %a
log CFU/ml %a
L. plantarum EM 9.780.09a
8.090.50b 82.7
8.520.07b 87.1
5.531.20c 56.5
All values are means standard deviation. 483 a % Survival: final
(CFU/ml)/control (CFU/ml) 100. 100% survival indicates that the
growth rate of the strain was not affected by the treatment. 484 b
PBS : Phosphate-buffered saline. 485 c SGJ : Simulated gastric
juice. 486 * Means in the same row with different superscript
letters are significantly different (p < 0.05) by Duncans
multiple range test. 487
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Table 3 488 Minimum inhibitory concentrations of antibiotics fo
r L. plantarum EM. 489
a Breakpoints are according to the guidelines of the EFSA (EFSA
2012). 490 b Strains with MICs lower than or equal to the
breakpoints are considered susceptible. AMP: ampicillin; 491 VAN:
vancomycin; GEN: gentamycin; KAN: kanamycin; STR: streptomycin,
ERY: erythromycin; CLI: 492 clindamycin; TET: tetracycline; CHL:
chloramphenicol. 493 c n.r. Not required.494
Strain MIC (g/ ml)b
AMP VAN GEN KAN STR ERY CLI TET CHL
L. plantarum EM 2 >512 0.5 16 4 0.125 1 16 4
Break points for L. plantaruma 2 n.r.c 16 64 n.r. 1 2 32 8
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Table 4 495 Antimicrobial activity of L. plantarum EM. 496
Antimicrobial activity was measured by the spot-on-the-lawn
test.497
Microorganisms Sensitive strains Antimicrobial
activity (AU/mL)
Gram-positive bacteria
Bacillus cereus KCTC 3624 3,100
Micrococcus luteus ATCC 15307 800
Staphylococcus aureus ATCC 29123 200
Gram-negative bacteria
E. coli O157:H ATCC 43895 1,500
Pseudomonas aeruginosa ATCC 27853 3,100
Salmonella enterica serovar Typhi ATCC 19430 3,200
Vibrio parahaemolyticus ATCC 17802 25,600
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Table 5 498 Growth of lactic acid bacteria in the presence of
bile acid. 499
All values are means standard deviation. 500 Means in the same
column for a single strain with different lowercase superscript
letters are significantly 501 different (p < 0.05) by Duncans
multiple range test. Means in the same row with different uppercase
502 superscript letters are significantly different (p < 0.05)
by Duncans multiple range test.503
Media
A600
L. plantarum EM
L. sakei DC1
L. acidophilus ATCC 43121
MRS 10.470.01aA 5.750.02aC 9.870.01aB
MRS+0.3% TDCA+0.1 g/L water-soluble cholesterol 9.740.01bA
4.980.07bC 9.180.09bB
MRS+0.5% TDCA+0.1 g/L water-soluble cholesterol 9.040.01cA
4.910.02bcB 9.090.09bA
MRS+0.3% Oxgall+0.1 g/L water-soluble cholesterol 8.570.03dA
4.830.04bcB 8.490.11cA
MRS+0.5% Oxgall+0.1 g/L water-soluble cholesterol 7.960.06eB
4.740.20cC 8.480.11cA
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Table 6 504 Cholesterol removal by growing, resting, and dead
cells of LAB. 505
Strains Cell state
0.5% oxgall
0.5% TDCA
Cholesterol removal (%)
Cholesterol removal (%)
L. plantarum EM
Growing cell 88.122.80a
47.660.00b
Resting cell 60.590.83c
38.200.00c
Dead cell 39.020.00d
33.330.00d
L. sakei DC1
Growing cell 6.442.10g
6.381.20f
Resting cell 5.292.50g
5.900.40f
Dead cell 1.220.00h
0.000.00g
L. acidophilus ATCC 43121
Growing cell 80.690.70b
51.700.30a
Resting cell 32.350.83e
15.450.40e
Dead cell 29.270.00f
15.150.00e
Growing, resting, and dead cells were incubated at 37 C for 24 h
in 0.5% oxgall- as well as 0.5% TDCA-506 containing cholesterol
(0.1 g/l) media. 507 * Means in the same column with different
superscript letters are significantly different (p < 0.05) by
508 Duncans multiple range test. 509
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Figure captions 510 511 Fig. 1. Cholesterol removal by cell wall
fractions of L. plantarum EM () and L. acidophilus ATCC 512
43121 (). 513 Fig. 2. Scanning electron micrographs of LAB in
media containing no cholesterol (A) and media 514 containing
cholesterol (B).515
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516 Fig. 1 517
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LAB Cell state
L. plantarum EM L. sakei DC1 L. acidophilus ATCC 43121
Growing cell
A
B
Dead cell
A
B
Cell wall fraction
A
B
Fig. 2 518
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Highlights
L. plantarum EM showed high cholesterol removal by growing and
even dead cells.
Cell wall fraction of L. plantarum EM removed cholesterol.
L. plantarum EM met the functional criteria required for
beneficial probiotics.
L. plantarum EM could be a promising probiotic candidate
regardless of its viability.
