Characterization of a potential probiotic bacterium lactobacillus plantarum ph04 with cholesterol lowering effect

Isolation of lactic acid bacteria with bile salt hydrolase activity .... Isolation and identification of lactic acid bacteria with bile salt hydrolase activity ... Recent studies show th

Thesis for the Degree of Master of Science

Characterization of a Potential Probiotic

Bacterium Lactobacillus plantarum PH04

with Cholesterol-Lowering Effect

Characterization of a Potential Probiotic

Bacterium Lactobacillus plantarum PH04

with Cholesterol-Lowering Effect

Advisor: Prof Myung Suk Lee

by

Thuy Duong Thi Nguyen

A thesis submitted in partial fulfillment of the requirements

for the degree of

Master of Science

In the Department of Microbiology, Graduate School

Pukyong National University

August 2005

Acknowledgements

First of all, I deeply appreciate the guidance, support and encouragement of my adviser, Professor Myung Suk Lee Throughout my studying in Microbiology Department, she has provided the best condition for my practice, as well as has broaden my knowledge by valuable discussions I thank Professor Young Tae Kim for useful comments on my thesis I extend my sincere gratitude to Dr Ji Hee Kang, who is willing not only to give continuous suggestions but also to support in solving technical problems

of my experiment I also acknowledge helpful topics of Professors Won Jae Lee, Tae Jin Choi and Young Hwan Song Special thanks are due to Professor Sang Bong Kim in Mechatronics Department for his motivation and encouragement For the same reason, I send my thanks to Dr Tan Tien Nguyen in Ho Chi Minh City University of Technology

I am indebted to my friends in Laboratory of General Microbiology, who enthusiastically assist me in preparation of this work, especially Young Ju Kim, Ji Sun Lee, Mi Ju Park, Kyung Taek Kim, Eun Mi Lee, Su Yeon Chang, Hae Suk Cho and So Yeon Han In addition, I would like to thank Mrs Young Sil Jeong in Central Laboratory for analysis assistance Thanks are also due to my Vietnamese friends for their contribution in various ways, especially Dr Thien Phuc Tran, Hoang Son Ngoc Tran, Nguyen Man Le, My Le Du and Le Duyen Thi Huynh

Finally, I am extremely grateful to my dear family: my parents, husbands’ parents, younger sisters Lan Anh Thi Nguyen and Hoang Anh Dang Phan, younger brothers Ngoc Huy Phung and Thanh Hai Le, and especially to my husband Chi My Phan and daughter Phuc An Nguyen Phan for their tremendous support and love

Thuy Duong Thi Nguyen

August, 2005

Acknowledgements vii

List of tables x

List of figures xi

Abstract xii

Introduction 1

Materials and methods 7

1 Isolation of lactic acid bacteria with bile salt hydrolase activity 7

1.1 Sample 7

1.2 Isolation of lactic acid bacteria with bile salt hydrolase activity 7

2 Identification of isolated strain 8

2.1 Physiological and biochemical characterization 8

2.2 Scanning electron microscopy 8

2.3 SDS-PAGE of whole cell protein 9

2.4 Sequencing of 16S rRNA 9

3 Characterization of isolated strain 10

3.1 Bile/ acid tolerance 10

3.2 Bile salt hydrolase activity 10

3.3 Growth phase regulation 11

3.4 Induction of bile salt hydrolase activity by bile salts 11

3.5 Effect of growth condition on bile salt hydrolase activity 12

3.6 Production of toxic substances 12

3.7 Antibiotics susceptibility 13

4 In vivo trial with mice for cholesterol-lowering effect 13

4.1 Preparation of bacterial suspension 13

4.2 Mice and diet 14

4.3 Monitoring mice 14

4.4 Serum total cholesterol and triglycerides 14

4.5 Bacterial translocation 15

4.6 Statistical analysis 15

Results 16

1 Isolation and identification of lactic acid bacteria with bile salt hydrolase activity 16

1.1 Isolation of lactic acid bacteria with bile salt hydrolase activity 16

1.2 Identification of isolated strain 16

2 Characterization of Lactobacillus plantarum PH04 24

2.1 Bile/acid tolerance 24

2.2 Growth phase regulation 24

2.3 Induction of bile salt hydrolase activity by bile salts 24

2.4 Effect of growth condition on bile salt hydrolase production 29

2.5 Antibiotic susceptibility and incidence of toxic substances 29

3 In vivo trial with mice for cholesterol-lowering effect 37

3.1 Effect of feeding L plantarum PH04 on general health and fecal lactic acid bacteria 37

3.2 Effect of feeding L plantarum PH04 on cholesterol and triglycerides 37

3.3 Effect of feeding L plantarum PH04 on visceral weight index and bacterial translocation 37

Discussion 43

Summary (in Korean) 48

References 50

List of tables

Table 1 Carbohydrate fermentation pattern of PH04 by API 50CHL Kit 20

Table 2 Bile salt tolerance of L plantarum PH04 25

Table 3 Acid tolerance of L plantarum PH04 26

Table4 Effect of temperature on bile salt hydrolase production 30

Table 5 Effect of initial pH on bile salt hydrolase production by L plantarum PH04 31

Table 6 Effect of NaCl on bile salt hydrolase production by L plantarum PH04 32

Table 7 Effect of nitrogen source on bile salt hydrolase production 33

Table 8 Effect of carbon source on bile salt hydrolase production 34

Table 9 Antibiotic susceptibility of L plantarum PH04 35

Table 10 Production of toxic substances by L plantarum PH04 36

Table 11 Effect of feeding L plantarum PH04 on body weight 38

Table 12 Effect of feeding L plantarum PH04 on fecal lactic acid bacteria counts 39

Table 13 Effect of feeding L plantarum PH04 on serum cholesterol and triglycerides 40 Table 14 Effect of feeding L plantarum PH04 on visceral weight index of mice 41

Table 15 Effect of feeding L plantarum PH04 on bacterial translocation to organs 42

List of figures

Fig 1 Death in 2000 attributable to selected leading risk factor 2

Fig 2 Biosynthesis and degradation of bile acids 3

Fig 3 Bile salt hydrolase plate-assay on MRS agar supplemented with TDCA 17

Fig 4 Gram staining and morphology of PH04, colony form of PH04 on MRS plate 18

Fig 5 Scanning electron microscope of isolated strain PH04 19

Fig 6 Effect of pH, temperature and NaCl on growth of isolated strain PH04 21

Fig 7 SDS-PAGE profile of whole cell proteins of isolated strain PH04 22

Fig 8 Partial sequence of 16S rRNA of isolated strain PH04 23

Fig 9 Growth phase regulation of L plantarum PH04 27

Fig 10 Induction of bile salt hydrolase activity of L plantarum PH04 by bile salts 28

Characterization of a Potential Probiotic Bacterium

Lactobacillus plantarum PH04 with Cholesterol-Lowering Effect

Thuy Duong Thi Nguyen

Department of Microbiology, Graduate School,

Pukyong National University

Abstract

Hypercholesterol is a major risk factor in coronary heart disease, which is the main cause of death in many countries Current treatments for elevated blood cholesterol are drug therapy, however are often sub-optimal and carry a risk of side effects, therefore alternative therapies are continuously promoted to overcome the challenges Recent studies show that administration of some strains of lactic acid bacteria can reduce the serum cholesterol, partly due to the bile salt hydrolase (BSH) activity of the bacteria

In this study, the isolation and characterization of lactic acid bacteria with

bile salt hydrolase activity and evaluation of this strain in an in vivo trial as a

potential probiotic with cholesterol-lowering effect was accomplished

The criteria for a probiotic strain screening including host specificity (isolation from newborn baby feces), acid/bile tolerance, and bile salt hydrolase were applied to isolate one colony with the highest activity The isolate was identified by physiological and biochemical characteristics, total protein profile

and 16S rRNA sequencing for confirming it belonged to Lactobacillus plantarum group, and then it was designed as Lactobacillus plantarum PH04

Characterization of strain including growth characteristics (temperature 37oC, NaCl<6%, pH 6-11), acid and bile tolerance, antibiotics susceptibility, production

of toxic substances were determined The optimum condition for growth and

concentration (0-2%), initial pH (6.0-7.0), nitrogen source (polypeptone, bacto peptone, bacto yeast extract) and carbon source (glucose, mannose) BSH

activity of L plantarum PH04 was regulated by stationary phase, and was

induced to increase 1.3-1.8 fold by 0.4 mM of conjugated bile salts, but not induced by deconjugated bile salts

The in vivo trial was conducted with induced hypercholesterol mice for

investigating the lowering effect of cholesterol and safety of the strain With the dose of 107 CFU/mice per day, throughout experiment period of 14 days, the serum cholesterol and triglycerides in bacteria-feeding group decreased 7% and 10%, respectively, in correlation with the increase of total lactic acid bacteria of

1 log CFU/g compared with non-feeding bacteria group Administration of

L plantarum PH04 did not cause any significant differences of behavior, weight

gain, visceral organ index and bacterial translocation at different tissues

With the characteristics of bile salt hydrolase, acid/bile tolerance, having

no toxic metabolism and resistance to antibiotics, L plantarum PH04 which

showed the effect of decreasing cholesterol level in mice trial without any significant side effects, proved to be a potential probiotic strain for cholesterol-lowering effect

Introduction

According to World Health Organization (WHO) estimates, 16.7 million people around the world die of cardiovascular diseases each year, amounts to one-third of all deaths globally Among that rates, 4.4 million deaths are due to high blood cholesterol, thus it amounts to 18% of strokes and 56% of global cardio heart disease (Atlas of Heart Disease and Stroke, WHO, Sept 2004) The risk factor of high cholesterol which leads to the second death rate, especially in the Eastern Mediterranean, Europe, and also in South East Asia countries (Fig 1) indicates the importance of determining method to reduce serum cholesterol

Cholesterol is a waxy substance that occurs naturally in all parts of the

body, it can be obtained from the diet or be synthesized de novo in the liver

From cholesterol, many other compounds are synthesized involving hormones,

vitamin D, and bile acids (Fielding et al., 1985) The process of synthesizing bile

acids from cholesterol is regulated by bile acid feedback as shown in Fig 2

(Fears et al., 1986) A small fraction of the bile salts, approximately 500 mg/d

escapes absorption, eliminated in the feces, and thus activate the synthesis new bile salts from cholesterol Though this is a very small amount, it nonetheless represents a major pathway for the elimination of cholesterol

Fig 1 Death in 2000 attributable to selected leading risk factor Africa, AFR;

America, AMR; Eastern Mediteranean, EMR; Europe, EUR; South-East Asia, SEAR, ; Western Pacific, WPR (World Health Organiztion Resource, 2002)

Fig 2 Biosynthesis and degradation of bile acids (Fears et al., 1986)

Cholesterol travels in the blood in packages called lipoproteins Low density lipoproteins (LDL) carry most of the cholesterol in the blood, and is the main ingredient causing the form of plaque, which decreases oxygen and nutrient supply to the heart, and thus leads to the stroke or cardio heart disease High density lipoprotein (HDL) carry cholesterol from other parts of the body back to the liver, which leads to its removal from the body; thus HDL help keep cholesterol from building up in the walls of the arteries Triglyceride is a form of

fat carried through the blood stream (Brown et al., 1983)

For treating the hypercholesterol, several drugs are applied to block the formation of cholesterol at various stages in biosynthetic pathway Fungal inhibitors reduce LDL cholesterol in the gastrointestinal tract, resins prevent the reabsorption of bile salts by combining with them, thereby increasing their fecal loss Clofibrate and gemfibrozil diverts free fatty acids from the pathways of esterification into those of oxidation, thus decreasing the secretion of triacylglycerol and cholesterol containing LDL by the liver Although these drugs effectively reduce cholesterol levels, they are known to have side effects such as gastrointestinal problem, blood vessels affection, and difficulty in drug

combination therapy (Kane et al., 1996)

Recently lactic acid bacteria (LAB) have been attracted considerable

attention as a potential alternative treatment (Chandan, 1999; Roos et al., 2000)

Lactic acid bacteria are normal components of intestinal microflora, and relate to various health-promoting effects including inhibition of pathogenic

microorganisms (Sookkhee et al., 2001), improvement of immune system (Ozawa

et al., 1983, Fernandes et al., 1990), antimutagenic and anticarcinogenic activity

(Gilliland et al., 1989)

One of the important beneficial health effects of LAB is the reduction effect

in serum cholesterol which has been reported in some in vitro as well as in vivo

activity of the bile salt hydrolase (Tahri et al., 1996; Taranto et al., 1997; Dora et

al., 2002) Klaver et al (1993) reported that the removal of cholesterol from

media by Lactobacillus acidophillus was due to the discruption of the cholesterol

micelles caused by bile salt deconjugation and precipitation of cholesterol with the free bile salts as the pH of the media dropped from acid production during

growth Both L acidophillus and L casei deconjugate bile salts during growth by producing the enzyme bile salt hydrolase (Corzo et al., 1999; Dashkevicz et al., 1989) Another study by Noh et al (1997) revealed that L acidophillus

incorporated some of the cholesterol into cellular membrane during growth

In other in vivo trials, enhanced BSH activity would increase cholesterol

excretion as suggested by the results of several human and animal studies

(Anderson et al., 1999; Taranto et al., 2000; Kawase et al., 2000; Haberer et al.,

2003) The cholesterol-lowering potential of some strains have been studied

including Bifidobacterium longum (Xiao et al., 2003), L gasseri (Usman et al., 2000), L reuteri (Taranto et al., 2000), L acidophillus (De Rodas et al., 1996)

They suggested that due to the properties differences between conjugated and

deconjugated bile salts (Gilliland et al., 1990; De Smet et al., 1994;

De Rodas et al., 1996), LAB with deconjugation activity could be effective in

cholesterol reduction either by enhancing release of fecal bile salts, consequently

cholesterol is utilized to a greater extent for de novo synthesis of bile salts; or by

decreasing the solubility of cholesterol in the guts It has also been suggested that BSH should be a requirement in the selection of probiotic organism with cholesterol-lowering properties, as non-deconjugated organisms do not appear to

be able to remove cholesterol from the culture medium to any significant extent

(Tahri et al., 1996; 1997)

Since a reduction of 1% in serum cholesterol is associated with a 2 to 3%

reduction in estimated risk of coronary disease (Manson et al., 1992), the

application of an effective probiotic lactic acid bacteria strain is a promoting therapy for lowering cholesterol in replace of the current drugs which usually deal with side effects We therefore conducted this study to isolate and characterize a strain of lactic acid bacteria with bile salt hydrolase activity, and

evaluate it in an in vivo trial as a potential probiotic for cholesterol-lowering

effect The study was divided into three parts: isolation and identification of strain from the feces of newborn baby with bile/acid tolerance and high bile salt hydrolase, characterization of isolated strain, and evaluation of potential

probiotic of isolated strain in an in vivo trial with mice for

cholesterol-lowering effect

Materials and methods

1 Isolation of lactic acid bacteria with bile salt hydrolase activity

1.1 Sample

Lactic acid bacteria were isolated from feces of newborn babies which were obtained from Ilsin Christian Hospital (Busan, Korea) from September to December 2003

1.2 Isolation of lactic acid bacteria with bile salt hydrolase activity

Screening of strains from feces for stability to bile and acidic environment was accomplished according to the method of Gorbach et al (1989) Serial dilutions of the feces were made and spread onto LBS agar (Merck) supplemented with 0.15% (w/v) oxgall (Merck) The plates were incubated in microaerobic condition (10% CO2) at 37oC for 48 h The bacteria which proliferate on the plates were screened for stability to more acidic conditions using MRS broth (Difco Laboratories, Detroit, USA) with pH adjusted to 3.0 After incubation under microaerobic condition for 24 h, strains that showed the growth > 107 CFU/ml were selected for screening of bile salt hydrolase (BSH) activity

Selection of strains with BSH was tested through the plate assay of

Dashkevitz et al (1989) Plates were prepared by adding 0.5% (w/v) of sodium

salt of taurodeoxycholic acid (TDCA) (Sigma, St Louis, USA) and 0.37 g/l CaCl2

to MRS agar After autoclaving and solidifying, the plates were placed in microaerobic incubator for at least 48 h before use All plates were inoculated using an overnight MRS culture broth (30 µl), and then incubated in microaerobic at 37oC for 72 h Subsequently, the colony which showed the biggest precipitate zone was chosen and named PH04 for identification and further characterization

2 Identification of isolated strain

2.1 Physiological and biochemical characterization

Initial identification of isolated strain PH04 based on Gram staining, morphology and catalase reaction Carbohydrate fermentation pattern was tested by API 50CHL Kit (BioMereux, France) Effects of growth condition (pH 2-

11, temprature 5-60oC, NaCl concentration 0-10%) were assessed by inoculating 1% PH04 into MRS broth and incubating at 37oC for 24 h, then OD at 650 nm was measured

2.2 Scanning electron microscopy

Preparation of the PH04 cells for scanning electron microscopy (SEM) was

performed as described by Yamauchi et al (2000) The cells collected after

centrifugation (8,000 ×g, 10 min) were fixed in 2.5% (v/v) glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4), post fixed with 4% osmium tetroxide

in the same buffer for 2 h, washed twice with buffer, and dehydrated in 20, 40,

60, 80, 90 and 100% ethanol Then, the specimens for observation were subjected to critical-point drying, coated with gold and examined with Hitachi S-

2400 Scanning Electron Microscope (Japan)

2.3 SDS-PAGE of whole cell protein

Whole-cell proteins obtained by SDS-PAGE were analyzed by the modified

method of Pot et al (1993) Five milliliters of MRS culture broth (37oC, 18 h) of each strain was centrifuged (12,000 ×g, 3 min, 4oC) The cell pellet was washed twice with deionized water and suspended in 50 µl of Tris-HCl buffer (50 mM,

pH 8.0) Approximately 50 mg of glass beads (diameter, 425-600 µm; Sigma, St Louis, USA) was added to the tubes and vortexed for 5 min The tubes were heated for 5 min at 95oC, centrifuged and then analyzed by 12% SDS-PAGE Protein pattern of PH04 was compared with those of reference strains including

Lactobacillus plantarum L2-1 (Pasteur Institute, France), Lactobacillus casei, Lactobacillus acidophilus (Laboratory collection)

2.4 Sequencing of 16S rRNA

Polymerase chain reaction (PCR) of 16S rRNA was performed from total DNA of PH04 in thermal cycler MyGenie 32 (Bioneer, Korea) Total DNA of bacteria was extracted with AccuPrep Genomic Extraction Kit (Bioneer, Korea) Two primers for PCR were the sequences consisting degenerate bases that bind

to the conserved bases of 16S rRNA in eubacteria, nucleotides 49-68 and

1510-1492 of Escherichia coli 16S rRNA, i.e forward primer and reverse primer are

5’- AGAATTCTNANACATGCAAGTCGAIGC-3’,

5’-GTGGATCCGGYTACCTTGTTAACGACCTT-3’, respectively PCR products were purified using AccuPrep PCR purification Kit (Bioneer, Korea) and sequenced by ABI Prism 3700 DNA Sequencer (Applied Biosystem, Foster, CA)

3 Characterization of isolated strain

3.1 Bile/ acid tolerance

Bile/acid tolerance of strain was accomplished by measuring its growth or viability in the medium with either bile salts or low pH adjusting Bile salt tolerance was assessed by inoculating 1% (v/v) of isolate into MRS broth containing 0, 0.2 or 0.4% (w/v) oxgall (Difco), incubating culture broth in microaerobic condition at 37oC for 9 h Acid tolerance was determined by inoculating 10% (v/v) of strain into MRS broth previously adjusted to pH 2.0 by HCl, then the mixture was incubated at 37oC for 2 h Viable cell count was determined by pour plate method with MRS agar

3.2 Bile salt hydrolase activity

Bile salt hydrolase activity of whole cell was determined by measuring the

amount of amino acid liberated from conjugated bile salts (Tanaka et al., 2000)

From determined volume of culture broth (37oC, 24 h), cells were harvested by centrifugation (6,000 ×g, 10 min), washed twice with 0.1 M phosphate buffer (pH 6.0) and resuspended in the same buffer to reach the OD 3.0 at 600 nm The reaction mixture consisted of 350 µl of 0.1 M phosphate buffer (pH 6.0),

50 µl of 200 mM sodium salt of taurocholic acid (Sigma, St.Louis, USA) and

200 µl of washed cell suspension The tube was incubated at 37oC, and at defined time, sample (200 µl) was taken and 10 µl of 6 N HCl was added to terminate the reaction The sample was centrifuged (8,000 ×g, 5 min), the supernatant was collected for subsequently assaying for free amino group by

ninhydrin reaction To 100 µl of supernatant was added 1.9 ml ninhydrin reagent, the mixture was thoroughly mixed, boiled for 14 min, cooled for 3 min, then the absorbance at 570 nm was measured Ninhydrin reagent was prepared

by mixing 0.5 ml of 1% (w/v) of ninhydrin in 0.5 M citrate buffer (pH 5.5), 1.2 ml glycerol, and 0.2 ml of 0.5 M citrate buffer (pH 5.5) Specific unit of BSH activity (U/ml) was defined as the amount (nanomoles) of taurine released from the substrate for 1 minute from 1 ml of culture broth

3.3 Growth phase regulation

Effect of growth phase of PH04 on BSH activity was determined by inoculating 1% (v/v) PH04 in MRS broth, incubated at 37oC for 24 h At interval time of 3 h, the broth was withdrawed for determining pH, growth and BSH activity The growth of strain was determined by pour plate method with MRS agar

3.4 Induction of bile salt hydrolase activity by bile salts

Materials for testing the induction of BSH obtained from Sigma including sodium salts of taurocholic acid (TC), glycocholic acid (GC), taurodeoxycholic acid (TDCA), glycodeoxycholic acid (GDCA), cholic acid, taurine and glycine The culture broth (1%) of isolated strain was incubated to stationary phase (37oC,

12 h), then divided into portions, to each portion was added 0.4 mM of sole induction tested material, the control without addition All broth portions were continuously incubated at 37oC for 30 min, then the cells were withdrawed and tested for BSH activity

3.5 Effect of growth condition on bile salt hydrolase activity

Effects of growth condition on BSH activity of L plantarum PH04

including initial pH (2-11), temperature (5-60oC), NaCl concentration (0-10%), carbon and nitrogen sources were performed by inoculating 1% (v/v) of PH04 into MRS broth (initial pH 6.3), incubating for 24 h, and then measuring the OD

of culture broth at 650 nm, final pH and BSH activity

Carbon sources were investigated by substituting dextrose in MRS broth

by 20 g/l of the sole carbon source from either lactose, mannose, mannitol, fructose, galactose, sucrose, maltose or casein, respectively

Similarly, effect of nitrogen sources on BSH was investigated by substituting the mixture of nitrogen source (Bacto proteose peptone, Bacto beef extract, Bacto yeast extract) in MRS broth by 30 g/l of the sole nitrogen source from either ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium phosphate, potassium nitrate, polypeptone, proteose peptone, Bacto beef extract, Bacto yeast extract, Tryptone peptone, Bacto peptone or Phyto peptone

3.6 Production of toxic substances

Production of toxic substances by isolated strain including testing β-gelatin liquefaction, ammonia production, indole test, phenylalanine deaminase was performed as directions of MacFaddin (2000) β-hemolysin test was performed on 5.0% blood agar Gelatin liquefaction was determined by inoculation PH04 into nutrient gelatin stab medium, incubating at 37oC overnight and observing the turbidity and liquefaction Ammonia production was

tested by determining the ability of an organism to split urea into ammonia by the action of the urease; culture was inoculated in urea broth and the color change was observed after incubation at 37oC overnight Indole test was performed to determine the ability of strain to split indole from tryptophan using Ehrlich’s indole reagent Phenylalanine deaminase test aimed to examine the ability of strain to deaminate phenylalanine to phenylpyruvic acid, which known

as cancer inducer Production of β-glucuronidase was tested by API ZYM Kit (BioMereux, France)

3.7 Antibiotics susceptibility

Resistance against various antibiotics was tested by the disk diffusion assay The antimicrobial susceptibility test disks were obtained from BBL (Becton Dickinson and company, Cockeysville, USA) including erythromycine (E15), penicilline (P10), cefoxitin (CTT30), gentamicin (GM10), nalidixic acid (NA30), chloramphenicol (C30), ampicillin (AM10), tetracycline (TE30) and kanamycin (K30)

4 In vivo trial with mice for cholesterol-lowering effect

4.1 Preparation of bacterial suspension

centrifugation (6,000 ×g, 5 min), washed twice with sterile saline and then resuspended in 0.85% NaCl to the appropriate concentration

4.2 Mice and diet

Twelve ICR male mice obtained at the age of 5-6 weeks were housed in a room with controlled temperature and humidity They were fed a commercial

chow and water ad libitum for 6 days for adaptation, then were divided into two

groups of 6 each As a preliminary treatment to produce hypercholesterolemia,

the mice were fed the diet as described by Taranto et al (1998), which based on

10% (w/v) skim milk (Difco) supplemented with 10% cream (Frima, Korea) for 14 days For the next 14 days, the daily dose of 25 µl containing 107 CFU of

L plantarum PH04 was orally administered to one group, the other group

without feeding bacteria was used as a control

4.3 Monitoring mice

During the experiment, the animal activity, behavior and general health were monitored daily Feed intake, water intake were also recorded daily and body weight was measured weekly Fecal samples were counted for lactic acid bacteria before and after two weeks of bacteria administration by pour plate method using MRS agar, and the results were reported as CFU per gram of wet weight feces

4.4 Serum total cholesterol and triglycerides

After administration of bacteria for 14 days, the mice were euthanised by ethyl ether Blood was obtained from arteria cervicalis and the mice were subsequently autopsied for testing bacterial translocation Serum samples were analyzed for total cholesterol and triglycerides by Express Plus Analyzer (CHZRON Diagnostics, USA)

4.5 Bacterial translocation

Translocation of bacteria to blood and tissues was accomplished as the

method of Zhou et al (2000) Before excising tissue sample, surface of viscera

was swiped with a sterile swab that was subsequently spread on MRS plates to test the contamination of viscera The mesenteric lymph nodes (MLN), spleen and liver were excised aseptically and were collected into 0.2 ml Brain Heart Infusion broth (Difco), in which the tissues were homogenized with grinders (Sigma) The tissue suspensions (100 µl) and blood samples (15 µl) were plated

on MRS agar plates, then the plates were incubated at 37oC for 72 h under microaerobic condition The positive result was defined by the presence of colony

on the plate

4.6 Statistical analysis

Experimental data are presented as the mean and standard errors of the mean Paired-samples t-test was conducted with Microsoft Excel and SPSS 11.5 (SPSS Inc., Chicago, USA) to determine the statistical difference significance between two groups of bacteria-feeding and non bacteria-feeding The significance difference was set at P < 0.05

Results

1 Isolation and identification of lactic acid bacteria with bile salt hydrolase activity

1.1 Isolation of lactic acid bacteria with bile salt hydrolase activity

Among 123 colonies with bile/acid tolerance, one colony which showed the biggest precipitated zone by plate-assay (Fig 3) was selected and named PH04

1.2 Identification of isolated strain

Physiological and biochemical characteristics of PH04 were tested as in Bergey’s Manual of Determinative Bacteriology Gram staining, morphology and colony form on MRS plate were showed in Fig 4 Scanning electron microscopy

of PH04 was showed in Fig 5 Carbohydrate fermentation pattern by API 50CHL was summarized in Table 1 Growth condition characteristics of PH04 (Fig 6) were determined including initial pH 6-11, temperature 25-45oC, NaCl 0-6% SDS-PAGE of whole-cell protein of PH04 showed similarity with that of

Lactobacillus plantarum L2-1 (Pasteur, France) (Fig 7) Sequencing of 16S rRNA

of PH04 (Fig 8) showed 97% similarity with Lactobacillus plantarum LP3

(GenBank accession No AY675256) Therefore, the isolated strain was

designated as Lactobacillus plantarum PH04

(D)

(C) (B)

(A)

Fig 3 Bile salt hydrolase plate-assay on MRS agar supplemented with TDCA

Isolated strain PH04, (A); L acidophilus, (B); L casei, (C); B lactis, (D)

Fig 4 Gram staining and morphology of PH04, (A); colony form of PH04 on MRS

plate, (B)

Fig 5 Scanning electron microscope of isolated strain PH04

Table 1 Carbohydrate fermentation pattern of PH04 by API 50CHL Kit

No Active ingredients Lactobacillus

plantarum

Isolated strain

No Active ingredients Lactobacillus

plantarum

Isolated strain

0 0.5 1 1.5 2 2.5 3

0 2 4 6 8 10

NaCl (%)

Fig 6 Effect of pH, (A); temperature, (B); and NaCl concentration, (C) on growth

of isolated strain PH04 Inoculation of 1% PH04 in MRS broth at 37oC for

24 h

(A) (B) (C) (D)

Fig 7 SDS-PAGE profile of whole cell proteins of isolated strain PH04, (A);

L plantarum L2-1, (B); L casei, (C); L acidophilus, (D)