Accepted Manuscript

Cholesterol-lowering effects of a putative probiotic strain Lactobacillus plantarum EM

isolated 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 probiotic
strain Lactobacillus plantarum EM isolated from kimchi, LWT - Food Science and Technology (2015),
doi: 10.1016/j.lwt.2015.01.019.
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Cholesterol-lowering effects of a putative probiotic strain

Lactobacillus plantarum EM isolated from kimchi

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Eun A Choi Hae Choon Chang*

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Department of Food and Nutrition, Kimchi Research Center, Chosun University, 309

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Pilmun-daero, Dong-gu, Gwangju 501-759, South Korea

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e-mail: hcchang@chosun.ac.kr

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Corresponding author: Tel.: +82 62 2307345; fax: +82 62 2228086

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Abstract

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Lactobacillus plantarum EM, which was isolated from kimchi, showed high cholesterol removal by

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growing, resting, and even dead cells. Moreover, cell wall fraction of L. plantarum EM removed

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cholesterol in a cell wall concentration-dependent pattern. Lactobacillus acidophilus ATCC 43121 as a

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control showed high cholesterol removal by growing cells, whereas resting and dead cells showed less

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cholesterol removal. Scanning electron micrographs showed that large amounts of cholesterol adhered to

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surfaces of growing and dead cells of L. plantarum EM without changes in cell morphology. On the other

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hand, only a small amount of cholesterol adhered to the surface of L. acidophilus ATCC 43121 cells, and

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growing cells of L. acidophilus ATCC 43121 showed morphological changes with a thinner and longer

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shape. Based on these results, cholesterol removal mechanisms by L. plantarum EM could be attributed to

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enzymatic assimilation including bile salt hydrolase activity and cell surface-binding. Further, L.

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plantarum EM showed reasonable tolerance to acid and bile stresses and displayed antagonistic activity

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against pathogens. Moreover, L. plantarum EM did not represent a health risk due to antibiotic resistance.

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The results of this study indicate that L. plantarum EM may be a promising probiotic candidate and

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adjunct culture for reduction of serum cholesterol level regardless of its viability.

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Keywords: Cholesterol-lowering effect, Lactobacillus plantarum, Probiotic, Kimchi

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1. Introduction

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Lactic acid bacteria (LAB) are industrially important microorganisms worldwide for the fermentation

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of foods. Moreover, LAB as probiotics have become the focus of intensive international research for their

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health-promotion effects, which include reduction of serum cholesterol level, stimulation of immune

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responses, cancer prevention, and alleviation of diarrhea (De Vrese & Schrezenmeir, 2008). The

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numerous health benefits of LAB have made them into promising probiotic candidates that are studied for

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their desirable properties. The World Health Organization (WHO) has predicted that cardiovascular

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disease will remain the leading cause of death through 2030, affecting approximately 23.6 million people

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worldwide (WHO, 2009). Increased serum cholesterol correlates highly with coronary heart disease

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(Kumar et al., 2012). Thus, investigators are paying close attention to the cholesterol-lowering effects of

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LAB among their many functional effects.

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Studies have indicated that several LAB strains, mainly Lactobacillus spp., have cholesterol-reducing

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effects in vitro or in vivo (Kumar et al., 2012; Ooi & Liong, 2010). However, the exact mechanisms of

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serum cholesterol reduction by probiotic bacteria are not completely clear. Several mechanisms have been

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proposed in vitro, including assimilation (Pereira & Gibson, 2002), surface binding (Liong & Shah, 2005),

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incorporation into cellular membranes (Lye, Rusul, & Liong, 2010a), co-precipitation with deconjugated

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bile (Liong & Shah, 2006), enzymatic deconjugation of bile acids by bile salt hydrolase (BSH) (Lambert,

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Bongers, de Vos, & Kleerebezem, 2008), conversion of cholesterol into coprostanol (Lye, Rusul, & Liong,

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2010b), and production of short-chain fatty acids by probiotics (De Preter et al., 2007).

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The aim of this study was to investigate high cholesterol removal by dead cells of the selected LAB

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strain as well as its possible mechanisms of action. Moreover, the selected strain was identified and

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evaluated in terms of its probiotic properties, such as acid and bile tolerance, antimicrobial activity

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against pathogens, and antibiotic resistance.

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2. Materials and methods

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2.1. Bacterial cultures and screening of LAB strains for cholesterol removal

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Screening of LAB strains from kimchi for cholesterol removal was carried out by determination of
BSH activity (Dashkevicz & Feighner, 1989) and cholesterol assimilation (Rudel & Morris, 1973).

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2.1.1. BSH activity

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For BSH activity measurement, 10 l of culture grown in MRS broth was spotted onto BSH screening

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medium, which consisted of de Man Rogosa and Sharpe (MRS; Difco, Sparks, MD, USA) agar

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supplemented with 0.5% (w/v) sodium salt taurodeoxycholic acid (TDCA, Sigma-Aldrich, St Louis, MO,

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USA) and 0.37 g/l of CaCl2 (Dashkevicz & Feighner, 1989). Plates were incubated anaerobically at 37 C

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using GasPak EZ anaerobe container systems (Becton, Dickinson and Company, Sparks, MD, USA),

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after which BSH activity was determined by measuring the diameters of precipitation zones. BSH activity

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was expressed based on the diameters of precipitation zones on BSH screening medium: -, no

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precipitation; +, precipitation zone of up to 10 mm; ++, precipitation zone of up to 15 mm; and +++,

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precipitation zone of up to 20 mm.

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2.1.2. Cholesterol assimilation

Cholesterol assimilation was determined using the method of Rudel and Morris (Rudel & Morris,

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1973). LAB cells grown overnight were inoculated (1%) and cultivated anaerobically using GasPak EZ

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anaerobe container systems (Becton, Dickinson and Company) at 37 C in MRS broth supplemented with

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0.5% (w/v) oxgall (Sigma-Aldrich) and 0.1 g/l of water-soluble cholesterol (Sigma-Aldrich). Following

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incubation, cells were harvested (9,950 g, 5 min, 4 C), after which 1 ml of supernatant was added to

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2 ml of 33% (w/v) potassium hydroxide and 3 ml of 95% (v/v) ethanol. The mixture was shaken well for

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1 min and then heated for 10 min in a 60 C water bath. After cooling in cold water, 5 ml of hexane was

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added, followed by mixing and addition of 1 ml of distilled water. The tube was allowed to stand for 10

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min at room temperature for phase separation, after which 3 ml of the hexane phase was transferred to a

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clean tube. The hexane phase was then evaporated under a nitrogen stream. The concentrated phase was

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added to 4 ml of freshly prepared O-phthalaldehyde (Sigma-Aldrich, 0.5 mg of O-phthalaldehyde/ml of

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acetic acid), mixed, and permitted to stand at room temperature for 10 min. Following the addition of

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2 ml of concentrated sulfuric acid and incubation for 10 min, the absorbance at 550 nm was read using a

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spectrophotometer (Amersham Biosciences, Uppsala, Sweden). Absorbance values were compared to

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those obtained using cholesterol standard.

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2.2. Identification of isolate

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The LAB strain showing the highest BSH activity and cholesterol assimilation during measurement of

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cholesterol removal was identified for further study. Identification was carried out by Gram staining,

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catalase test, and morphological observation under a microscope, and 16S rRNA gene sequences were

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determined using an API prism 3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA)

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according to the method described by Yang and Chang (Yang & Chang, 2008). The determined 16S rRNA

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gene sequences were compared with sequences available in the GenBank database using the Clustal W

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program (Thompson, Higgins, & Gibson, 1994).

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2.3. Growth in bile acids and cholesterol

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Growth of LAB under bile acids and cholesterol was determined. LAB grown overnight in MRS were

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inoculated (1%) into MRS broth supplemented with 0.3% (w/v) oxgall and 0.1 g/l of water-soluble

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cholesterol, 0.5% (w/v) oxgall and 0.1 g/l of water-soluble cholesterol, 0.3% (w/v) TDCA and 0.1

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g/l of water-soluble cholesterol, and 0.5% (w/v) TDCA and 0.1 g/l of water-soluble cholesterol.

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Growth of LAB in MRS broth without bile was used as a control. Initial pHs of the prepared media were

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pH 6.2-6.4. The culture was incubated anaerobically for 24 h at 37 C, after which the absorbance at 600

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nm was measured.

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2.4. Acid and bile tolerance

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Tolerance levels of LAB to acid and bile salts were determined as previously described (Ryu & Chang,
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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

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min, 4 C), after which cell pellets were resuspended in phosphate-buffered saline (PBS, pH 2.5; Hyclone,

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Logan, UT, USA), simulated gastric juice (SGJ; 3 mg of pepsin dissolved in 1 ml of 0.5% saline buffer,

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pH 2.5), or bile salt (0.3% oxgall dissolved in PBS, pH 8.0) with approximately 9.8 log CFU/ml of cell

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numbers. The suspensions were incubated at 37 C for 1 h in PBS or SGJ for 3 h in bile salt. Thereafter,

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suspensions were harvested (9,950 g, 5 min, 4 C) and resuspended in MRS broth, after which viable

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cell counts were enumerated on MRS agar after incubation at 37 C for 48 h. LAB suspended in MRS

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broth without acid or bile salt were considered as controls.

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2.5. Antibiotic susceptibility

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Antibiotic susceptibility of the selected LAB isolate was determined according to the technical

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guidelines of the European Food Safety Authority (EFSA) (EFSA, 2012) using a previously described

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method (Ryu & Chang, 2013). The minimal inhibitory concentrations (MIC) of antibiotics were

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measured. After culturing LAB in MRS broth for 24 h, cells were centrifuged and resuspended in Muller

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Hinton (MH; Difco) broth containing 0.5% dextrose. Resultant cell suspensions were diluted in the same

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medium to a cell density of 5.0 log CFU/ml (A600=0.4-0.5). Each antibiotic was added to the diluted cell

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suspension, which was incubated anaerobically at 37 C for 24-48 h. Cell growth was observed visually

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and measured based on absorbance at 600 nm (Amersham Biosciences). MIC values were defined as the

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lowest antibiotic concentrations where no bacterial growth occurred. All antibiotics were purchased from

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Sigma-Aldrich.

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2.6. Antimicrobial activity

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LAB grown overnight were inoculated (1%) into MRS broth and incubated anaerobically at 37 C for

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24 h. The culture was centrifuged, after which the supernatant was membrane-filtered (0.4 m membrane

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filter; Advantec MFS, Dublin, CA, USA) and then freeze-dried (Samwon, Busan, South Korea). The

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freeze-dried culture supernatant was concentrated (5-fold) in 20 mM sodium acetate (pH 4.0).

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Concentrated (5-fold) MRS broth was used as a control.

Antimicrobial activities against seven pathogens were investigated using spot-on-the-lawn assay

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(Hoover & Harlander, 1993). Pathogens were grown at 37 C on Luria-Bertani (LB; Difco) agar for

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E.coli, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhi, nutrient agar (NA; Difco)

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supplemented with 2% (w/v) NaCl for Vibrio parahaemolyticus, and tryptic soy agar (TSA; Difco) for

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Bacillus cereus, Micrococcus luteus, and Staphylococcus aureus. ATCC strains were purchased from the

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American Type Culture Collection (Manassas, VA, USA), whereas KCTC strains were purchased from

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the Korean Collection for Type Cultures (Daejeon, South Korea). LB, TSA, or NA plates were spread

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with each pathogen at a concentration of 6 log CFU/ml. An aliquot (10 l) of LAB culture was spotted

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onto sensitive pathogen plates for each antimicrobial sample. Antimicrobial activity, expressed as

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arbitrary units (AU) per milliliter, was defined as the reciprocal of the highest dilution at which pathogen

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growth was inhibited (Ammor, Tauveron, Dufour, & Cherallier, 2006). The antimicrobial titer was

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calculated as (1000/d) D, where D is the dilution factor and d is the dose (amount of antimicrobial

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samples pipetted onto each spot). The above experiment was performed in triplicate.

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2.7. Cholesterol removal by growing, resting, and dead cells

For cholesterol assimilation by growing cells, LAB cells were inoculated (1%) and grown

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anaerobically at 37 C in MRS broth supplemented with 0.5% (w/v) oxgall and 0.1 g/l of water-soluble

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cholesterol as well as in MRS broth containing 0.5% (w/v) TDCA and 0.1 g/l of water-soluble cholesterol.

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To prepare resting and dead cells (Lye, Rusul, & Liong, 2010a), LAB strains were incubated

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anaerobically at 37 C in MRS broth containing 0.5% oxgall as well as in MRS broth containing 0.5%

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TDCA. The cell pellet obtained from the media was washed twice with sterile-distilled water. For

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cholesterol removal by resting cells, the cell pellet was suspended in 0.05 M phosphate buffer (pH 6.8)

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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 of

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water-soluble cholesterol. For dead cell assay, the cell pellet was suspended in saline and heat-killed at

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121 C for 15 min. The dead cells were harvested, after which the pellet was suspended in MRS

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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

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water-soluble cholesterol.
All strains were incubated at 37 C for 24 h, after which the mixture was centrifuged (9,950 g, 5 min,

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4 C). The remaining cholesterol concentration of broth was determined using the method of Rudel and

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Morris (Rudel & Morris, 1973) described in 2.1.1. Cholesterol assimilation (Ooi & Liong, 2010) was

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expressed as {0.1 g/l of cholesterol (control) concentration of remaining cholesterol in cultures}/control

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100.

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2.8. Cholesterol removal by LAB cell wall

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2.8.1. Preparation of cell wall

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Isolation of LAB cell wall was carried out as previously described with modifications (Piuri, Sanchez-

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Rivas, & Ruzal, 2005). Exponential phase of LAB cultures (20 ml) was harvested, washed twice with

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PBS (pH 7.0-7.2, Hyclone), and suspended in 20 ml of PBS at 4 C. Ice-jacketed cell suspensions were

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sonicated (Amp 60, 10 min, pulse 2 sec; Sonics, Newton, CT, USA) twice, centrifuged, suspended in 5%

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(w/v) sodium dodecyl sulfate (SDS, Roche, Indianapolis, IN, USA), and then incubated at 60 C for 30

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min. The crude cell wall fraction was centrifuged (9,950 g, 30 min, 4 C), washed twice with PBS,

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suspended in 10 mM Tris-HCl (pH 7.5) with 10 mM MgCl2, and subsequently incubated with a mixture

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of 0.01 g/ml of DNase (Sigma-Aldrich), 0.01 mg/ml of RNase A (Sigma-Aldrich), and 0.01 g/ml of

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trypsin (Sigma-Aldrich) for 4 h at 37 C. Finally, the cell wall fraction was centrifuged, washed three

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times with PBS, suspended in 20 ml of PBS, and then homogenized twice by sonication (Amp 60, 10 min,

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pulse 2 sec). The purified cell wall fraction was then freeze-dried.

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2.8.2. Cholesterol removal by LAB cell wall fraction

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The prepared cell wall fraction was suspended in MRS containing 0.5% TDCA and 0.1 g/l of water-

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soluble cholesterol along with A600=1, 2, 3, 4, and 5 mg/ml of the LAB cell wall fraction. The suspension
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was incubated anaerobically at 37 C for 24 h, after which the mixture was centrifuged (9,950 g, 30 min,

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4 C). The remaining cholesterol concentration of the supernatant was measured using the method of

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Rudel and Morris (Rudel & Morris, 1973), and its cholesterol assimilation (%) was determined.

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2.9. Scanning electron microscopy (SEM)

Growing and dead cells as well as cell wall fraction were prepared as described in sections 2.7 and 2.8.

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The prepared growing and dead cells (A6008.0) as well as cell wall fraction (A60012.0) were suspended

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in MRS broth supplemented with 0.5% TDCA and 0.1 g/l of cholesterol incubated at 37 C for 24 h. The

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prepared LAB cultures in MRS broth without any supplementation were used as a control. After

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incubation, LAB cultures were centrifuged and then washed twice with PBS (pH 7.0-7.2, Hyclone).

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Cholesterol attached to LAB cells or cell wall fraction was observed using field emission scanning

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electron microscopy (FE-SEM; Hitachi, Tokyo, Japan) as described previously with slight modifications

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(Kalchayanand, Dunne, Sikes, & Ray, 2004). LAB cell or cell wall fraction was prefixed with 0.1 M

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sodium cacodylate buffer (pH 7.4) containing 2.5% (v/v) glutaldehyde (pH 7.4; Sigma-Aldrich) for 2 h at

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4 C, followed by washing three times with 0.1 M sodium cacodylate buffer (pH 7.4). The samples were

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then postfixed with 1% (w/v) osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.4) for 1-2 h,

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washed three times with 0.1 M sodium cacodylate buffer (pH 7.4), and dehydrated in an ascending

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ethanol series [(%, v/v): 50, 60, 70, 80, 90, 95, and 100 ethanol] for 2 min each. Finally, the samples were

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immersed two times in 100% tert-butanol for 20 min and then coated with platinum using a sputter coater

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(E-1030 Ion sputter, Hitachi, Tokyo, Japan). The resultant chips were viewed in a FE-SE microscope.

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2.10. Statistical analysis

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Data are presented as the means and standard deviations (means SD) of three independent

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experiments performed in duplicate or triplicate. Statistical analysis was performed on the data using

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Duncans Multiple Range Test (DMRT) by SPSS ver. 21.0 for Windows (SPSS, Chicago, IL, USA) with

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statistical significance determined at p < 0.05.

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3. Results and discussion

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3.1. Isolation and identification of cholesterol-lowering LAB

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Seventy LAB cultures were isolated from 28 kimchi samples collected from 17 cities in South Korea.

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All isolates were grown well at 30 C in MRS medium. However, only 50 LAB strains among the 70 total

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LAB strains could be grown at 37 C. These 50 LAB strains were preselected for further experimentation.

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BSH activity and cholesterol assimilation of the 50 preselected LAB strains were examined (Table 1). Of

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the 50 tested LAB strains, 16 LAB strains expressed BSH activity, and their cholesterol assimilation

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varied from 3.51-80.86%. Based on this result, we selected two strains (EM and DC1) for further

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experimentation. Among the 50 strains, EM showed the strongest cholesterol assimilation (80.86%) and

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BSH activity (17.2 mm precipitation zone), whereas DC1 showed the weakest cholesterol assimilation

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(3.51%) and BSH activity (5.3 mm precipitation zone).

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The EM and DC1 strains were Gram-positive, rod-shaped, and catalase-negative. When 16S rRNA

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gene sequences of the EM (1,881 bp) and DC1 (1,471 bp) isolates were determined and compared with

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those of type strains in GenBank, EM and DC1 sequences showed 99.9% and 99.9% homologies with

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those of L. plantarum JCM 1149T and L. sakei DSM 20017T, respectively. Thus, EM and DC1 isolates

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were finally designated as L. plantarum EM and Lactobacillus sakei DC1, respectively, and their gene

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sequences were deposited in GenBank (accession numbers KC 422389 for EM and KJ 812206 for DC1).

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3.2. Acid and bile tolerance

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It is well known that probiotic bacteria should be capable of surviving passage through the

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gastrointestinal tract based on acid tolerance to human gastric juice as well as bile tolerance for survival

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in the small intestine (Saarela, Mogensen, Fondn, Mtt, & Mattila-Sandholm, 2000). Therefore, these

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characteristics may serve as suitable criteria for probiotic bacteria selection. L. plantarum EM showed

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high tolerance to acidic conditions at pH 2.5 (PBS and SGJ) after 1 h of exposure at 37 C (Table 2).

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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

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counts (9.78 log CFU/ml) of L. plantarum EM decreased to 5.53 log CFU/ml. In this study, acid and bile

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treatments to L. plantarum EM were carried out under nutritional deficit. Considering the human

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gastrointestinal tract is full of food, the actual survival of L. plantarum EM in the gastrointestinal tract

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will actually be greater than the result shown in Table 2. This suggests that L. plantarum EM has

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reasonable tolerance to acid and bile and should be active in the gastrointestinal tract.

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3.3. Antibiotic resistance

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L. plantarum EM was assessed for its resistance to nine antibiotics, including those highlighted by the

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EFSA (Florez et al., 2006). As shown in Table 3, L. plantarum EM was susceptible to all of the antibiotics

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tested, except vancomycin. The technical guidelines of the EFSA have demonstrated that no breakpoints

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for vancomycin and streptomycin are required for L. plantarum (EFSA, 2012). Therefore, it seems

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reasonable to conclude that consumption of L. plantarum EM in this study does not represent a health risk

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to humans due to antibiotic resistance. It is recognized that probiotics lack undesirable properties such as

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antibiotic resistance (Saarela, Mogensen, Fondn, Mtt, & Mattila-Sandholm, 2000; Florez et al., 2006).

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Thus, any evaluation of new probiotic candidates, including those traditionally used in food fermentation,

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should carefully confirm their safety status.

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3.4. Antimicrobial activity

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L. plantarum EM showed strong activities against the seven tested pathogens (Table 4). In particular, L.

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plantarum EM showed its strongest activity against Vibrio parahaemolyticus ATCC 17802 (25,600 units)

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as well as its weakest activity against Staphylococcus aureus ATCC 29123 (200 units). L. plantarum EM

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showed a broad antimicrobial spectrum against Gram-positive and Gram-negative pathogens (Table 4).

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Antimicrobial substances produced from LAB are known to include H2O2, CO2, lactic and acetic acids,

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bacteriocin, and others (Usman & Hosono, 1999). However, most of these antimicrobial substances from

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LAB strains have a narrow antimicrobial spectrum, and thus their inhibitory effects are only against

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closely related species (Holzapfel, Geisen, & Schillinger, 1995). One of the most important functional
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requirements of probiotics is a broad antimicrobial spectrum as well as antagonism against pathogenic

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bacteria with strong antimicrobial activity (Saarela, Mogensen, Fondn, Mtt, & Mattila-Sandholm,

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2000). Thus, the results represented in Table 4 for L. plantarum EM fulfill the beneficial requirements of

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probiotics.

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3.5. Growth in the presence of bile acid

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Growth in the presence of bile acid (oxgall or TDCA) was examined using L. plantarum EM and L.

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sakei DC1 (Table 5). L. acidophilus ATCC 43121, which is characterized by high cholesterol assimilation

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(Gilliand & Walker, 1989; Gilliand, Nelson, & Maxwell, 1985), was used as a control strain. Generally,

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all three strains grew well in the presence of both bile acids. However, growth of the three LAB strains

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slightly decreased in TDCA- or oxgall-containing media. Oxgall showed a slightly higher toxic effect on

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LAB growth than TDCA. The antimicrobial action of bile is well known; bile salt at high concentrations

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can rapidly dissolve membrane lipids and cause dissociation of integral membrane proteins, resulting in

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leakage of cell contents and cell death (Begley, Gahan, & Hill, 2005). The results represented in Table 5

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suggest that the three LAB strains are moderately resistant to bile toxicity.

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3.6. Cholesterol removal

3.6.1. Cholesterol removal by growing, resting, and dead cells

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As shown in Table 6, cholesterol removal according to cell state was in the order of growing, resting,

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and dead cells. L. plantarum EM showed the highest cholesterol removal among the three tested strains

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regardless of cell state. As already shown in Table 5, L. sakei DC1 showed approximately 1/2 growth

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(A6005) compared to the other two LAB strains (A6008-10). However, cholesterol removal of L. sakei

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DC1 was much lower (1/3-1/30) than that of L. plantarum EM or L. acidophilus ATCC 43121 (Table 6).

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Cholesterol removal by growing cells of L. acidophilus ATCC 43121 was as high as that of L. plantarum

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EM. However, cholesterol removal by dead cells of L. acidophilus ATCC 43121 was significantly (p <
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0.05) lower than that of L. plantarum EM.

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3.6.2. Cholesterol removal by LAB cell wall fraction and SEM observation

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To determine the reason for such high cholesterol removal by dead cells of L. plantarum EM,

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cholesterol removal by cell wall fractions of both L. plantarum EM and L. acidophilus ATCC 43121 was

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investigated. As shown in Fig. 1, the cell wall fraction of L. plantarum EM removed cholesterol in a cell

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wall concentration-dependent pattern. On the other hand, the cell wall fraction of L. acidophilus ATCC

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43121 showed significantly (p < 0.05) lower cholesterol removal than that of L. plantarum EM. This

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result indicates that cholesterol can be removed by the cell wall fraction itself without any metabolic

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process.

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Thus, we tried to observe cholesterol attachment to LAB cells according to cell state (growing, dead

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cell, and cell wall fraction) using SEM. As shown in Fig. 2, large amounts of cholesterol adhered to the

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surfaces of growing and dead cells of L. plantarum EM. Adherence of cholesterol to the cell wall fraction

330

was observed only with L. plantarum EM, whereas no cholesterol adherence or alteration of cell

331

morphology could be observed with L. sakei DC1. On the other hand, only small amounts of cholesterol

332

adhered to surfaces of growing and dead cells of L. acidophilus ATCC 43121. Interestingly, L.

333

acidophilus ATCC 43121 cells underwent morphological changes in a growing state (Fig. 2 - growing cell

334

B), as evidenced by a longer and thinner cell shape (arrow indicated) compared to control growing cells

335

of L. acidophilus ATCC 43121 (Fig. 2 - growing cell A). Further, dead cells of L. acidophilus ATCC

336

43121 did not show any morphological changes. Noh et al. previously reported the incorporation of

337

cholesterol into the cellular membrane of L. acidophilus ATCC 43121 in the presence of oxgall and

338

cholesterol (Noh, Kim, & Gilliand, 1997). Namely, a portion of the assimilated cholesterol was recovered

339

in the membrane fractions of cells grown in media containing cholesterol, which suggests that cholesterol

340

may have altered the cellular membrane or wall of L. acidophilus ATCC 43121.

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Cholesterol removal (%) by growing cells was almost the same between L. plantarum EM and L.

342

acidophilus ATCC 43121 (Table 6). However, examination of cholesterol adherence onto these two LAB

343

using SEM showed that their configurations were quite different. These data indicate that the cholesterol

344

removal mechanism of L. plantarum EM is different from that of L. acidophilus ATCC 43121. As

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reported already by Noh et al. (Noh, Kim, & Gilliand, 1997), incorporation of cholesterol into the cellular

346

membrane of L. acidophilus ATCC 43121 during growth might result in morphological changes, as

347

represented in this study (Fig. 2). On the other hand, Hosono and Tano-oka reported cholesterol binding

348

to LAB varies among strains and species and hypothesized that these differences in binding abilities can

349

be attributed to chemical and structural properties of cell wall peptidoglycans containing amino acids

350

capable of binding to cholesterol (Hosono & Tano-oka, 1995). Thus, strong cholesterol attachment to L.

351

plantarum EM regardless of its cell state might be due to the unique in chemical and structural properties

352

of the L. plantarum EM cell wall compared to those of other LAB cell types. However, direct

353

investigation of the chemical and structural properties of the L. plantarum EM cell wall that promote

354

binding to cholesterol is required. Cholesterol removal by dead cells of LAB strains has been reported

355

previously, even though the degree of removal is significantly less compared to that of growing cells

356

(Zeng, Pan, & Guo, 2010; Liong & Shah, 2005; Mahdieh, Hamid, & Naheed, 2014; Sirilun, Chaiyasut,

357

Kantachote, & Luxananil, 2010; Emami & Bazargani, 2014). Further, cholesterol removal by dead cells

358

due to cholesterol attachment to the cellular membrane has been investigated based on scanning electron

359

microscopic observation of cholesterol binding to growing cells (Lye, Rusul, & Liong, 2010a; Ooi &

360

Liong, 2010). To the best of our knowledge, there is no direct evidence verifying cholesterol removal by

361

dead cells via binding of cholesterol to dead cells or the cell wall fraction itself.

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Based on the results in Table 6 and Fig. 2, the cholesterol-lowering effect of L. plantarum EM can be

363

considered to be a major part of enzymatic assimilation, including BSH activity. Further, L. plantarum

364

EM showed a cholesterol-lowering effect with strong binding capability to cell surfaces regardless of its

365

viability. Cholesterol bound to LAB strains has been shown to inhibit intestinal absorption of cholesterol

366

in previous investigations (Usman & Hosono, 1999). Thus, the high cholesterol-binding capability of L.

367

plantarum EM might be more effective in reducing the serum cholesterol level in humans. However, in

368

vivo evaluation of this strain in humans is required to assess the usage of probiotics for reducing serum

369

cholesterol.

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4. Conclusions

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373

As the term probiotics means for life, viable probiotics are required to have beneficial effects on their
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host. In this study, L. plantarum EM showed high cholesterol removal by growing, resting, and even dead

375

cells based on the high cholesterol-binding capacity of its cell wall fraction. Therefore, L. plantarum EM

376

could be a potent probiotic to reduce serum cholesterol regardless of its viability. Moreover, L. plantarum

377

EM also appeared to meet the functional criteria required for beneficial probiotics, such as acid and bile

378

tolerance, antagonistic activity against pathogens, and antibiotic susceptibility.

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Acknowledgment

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Agriculture, Food and Rural Affairs, South Korea.

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This research was supported by the Technology Development Program for Food, Ministry of

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Table 1
Cholesterol assimilation and BSH activities of LAB strains isolated from kimchi.
Strain

AB

rod

BC

coccus

5.95

BY

rod

8.33

C12

rod

10.54

C14

rod

15.50

CC

rod

8.33

CM1

short rod

8.92

CM3

short rod

33.78
11.94

CS

short rod

DC1

rod

3.51

11

DC2

rod

5.23

12

DC3

short rod

6.98

13

DJ1

coccus

14

DM1

coccus

15

DS

rod

+
-

SC

BSH activity

RI
PT

Shape

10

13.20

+
-

5.14

10.72

80.86

+++

53.83

EM

rod

GJ2

coccus

18

GJ6

short rod

13.29

19

GJ7

rod

17.70

20

GS

rod

7.07

21

HA1

short rod

22

HC

M
AN
U

16
17

8.78

coccus

10.54

77.61

+++

23

HD1

rod

24

HJ

short rod

10.72

25

JD

short rod

10.09

++
-

JH

short rod

8.92

JS

coccus

9.41

28

JT

coccus

10.81

29

JW

short rod

10.86

30

KO

short rod

7.39

31

KW

10.45

32

MA

33

MH

34

MO1

35
37

coccus

short rod

9.73

short rod

9.59

short rod

17.25

MP1

coccus

6.80

MS1

short rod

9.14

NG1

coccus

15.32

NJ1

short rod

4.91

39

NJ6

short rod

13.15

40

NM1

short rod

15.86

++

41

NO1

rod

78.11

++
+

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C

38

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D

26
27

36

478
479
480

Cholesterol
Assimilation (%)
11.71

No.

EP

476
477

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

*BSH activity was expressed based on the diameters of precipitation zones on BSH screening medium: -,
no precipitation; +, precipitation zone of up to 10 mm; ++, precipitation zone of up to 15 mm; and +++,
precipitation zone of up to 20 mm.
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Table 2
Acid and bile tolerance of L. plantarum EM.

SGJc

Survival after 3 h at pH 8.0

0.3% oxgall

log CFU/ml

log CFU/ml

%a

log CFU/ml

%a

log CFU/ml

%a

9.780.09a

8.090.50b

82.7

8.520.07b

87.1

5.531.20c

56.5

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All values are means standard deviation.

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.
b
PBS : Phosphate-buffered saline.
c
SGJ : Simulated gastric juice.
* Means in the same row with different superscript letters are significantly different (p < 0.05) by Duncans multiple range test.

EP

483
484
485
486
487

RI
PT

L. plantarum EM

PBSb

SC

Strain

Survival after 1 h at pH 2.5

Initial
mean counts

AC
C

481
482

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488
489

Table 3
Minimum inhibitory concentrations of antibiotics for L. plantarum EM.
MIC (g/ ml)b
Strain
GEN

KAN

STR

ERY

CLI

TET

CHL

L. plantarum EM

>512

0.5

16

0.125

16

Break points for L. plantaruma

n.r.c

16

64

n.r.

RI
PT

VAN

32

Breakpoints are according to the guidelines of the EFSA (EFSA 2012).

Strains with MICs lower than or equal to the breakpoints are considered susceptible. AMP: ampicillin;
VAN: vancomycin; GEN: gentamycin; KAN: kanamycin; STR: streptomycin, ERY: erythromycin; CLI:
clindamycin; TET: tetracycline; CHL: chloramphenicol.
c
n.r. Not required.

EP

TE
D

M
AN
U

SC

AC
C

490
491
492
493
494

AMP

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495
496

Table 4
Antimicrobial activity of L. plantarum EM.
Microorganisms

Antimicrobial
activity (AU/mL)

Sensitive strains
Bacillus cereus KCTC 3624
Micrococcus luteus ATCC 15307

800

Staphylococcus aureus ATCC 29123

E. coli O157:H ATCC 43895
Pseudomonas aeruginosa ATCC 27853

Salmonella enterica serovar Typhi ATCC 19430

Vibrio parahaemolyticus ATCC 17802

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Antimicrobial activity was measured by the spot-on-the-lawn test.

AC
C

497

23

200

1,500

SC

Gram-negative
bacteria

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Gram-positive
bacteria

3,100

3,100

3,200
25,600

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498
499

Table 5
Growth of lactic acid bacteria in the presence of bile acid.
A600
Media

L. plantarum
EM

L. acidophilus
ATCC 43121

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

SC

RI
PT

10.470.01aA

MRS

EP

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D

M
AN
U

All values are means standard deviation.

Means in the same column for a single strain with different lowercase superscript letters are significantly
different (p < 0.05) by Duncans multiple range test. Means in the same row with different uppercase
superscript letters are significantly different (p < 0.05) by Duncans multiple range test.

AC
C

500
501
502
503

L. sakei
DC1

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Table 6
Cholesterol removal by growing, resting, and dead cells of LAB.

Cholesterol
removal (%)

Cholesterol
removal (%)

Growing cell

88.122.80a

47.660.00b

Resting cell

60.590.83c

Dead cell

39.020.00d

Cell state

L. plantarum EM

L. sakei DC1

33.330.00d

6.442.10g

Resting cell

5.292.50g

Dead cell

1.220.00h

0.000.00g

80.690.70b

51.700.30a

32.350.83e

15.450.40e

29.270.00f

15.150.00e

Resting cell
Dead cell

6.381.20f
5.900.40f

EP

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D

Growing, resting, and dead cells were incubated at 37 C for 24 h in 0.5% oxgall- as well as 0.5% TDCAcontaining cholesterol (0.1 g/l) media.
* Means in the same column with different superscript letters are significantly different (p < 0.05) by
Duncans multiple range test.

AC
C

506
507
508
509

38.200.00c

Growing cell

Growing cell
L. acidophilus
ATCC 43121

RI
PT

0.5% TDCA

SC

Strains

0.5% oxgall

M
AN
U

504
505

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510

Figure captions

511
Fig. 1. Cholesterol removal by cell wall fractions of L. plantarum EM (
) and L. acidophilus ATCC

513
514
515

43121 (
).
Fig. 2. Scanning electron micrographs of LAB in media containing no cholesterol (A) and media
containing cholesterol (B).

AC
C

EP

TE
D

M
AN
U

SC

RI
PT

512

26

EP

TE
D

Fig. 1

AC
C

516
517

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SC

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LAB

L. plantarum EM

Cell state

L. sakei DC1

L. acidophilus ATCC 43121

RI
PT

Growing
cell

M
AN
U

SC

TE
D

Dead
cell

Cell wall
fraction

AC
C

EP

518

Fig. 2

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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.

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L. plantarum EM could be a promising probiotic candidate regardless of its viability.