指導老師 : 鄭伯智老師 林宏榮老師 指導教授 : 陳啟楨 speaker: 林季怡...

Functional properties of Lactobacillus strains isolated from kimchi

School of Life Sciences, Handong Global University, Pohang, Gyungbuk 791-708, Korea

指導老師 :鄭伯智老師 林宏榮老師指導教授 :陳啟楨Speaker: 林季怡報告日期 :100.05.22

Introduction

Kimchi is a Korean traditional fermented vegetable product and is gaining popularity as a functional food.

It is typically consumed in a raw (uncooked) state, and serves as a side-dish with practically every meal

The magazine Health mentioned kimchi in its list of the top five "World's Healthiest Foods"

Kimchi, being a lactic acid-fermented vegetable product consumed raw, is

therefore also considered to be a good source of potentially beneficial and useful LAB

Among these, particularly the lactobacilli are of interest as beneficial inhabitants of the intestinal tract of man and animals.

A wide range of such desired roles have been suggested for diverse strains of LAB.

Including immune stimulation, pathogen exclusion, production of bioactive substances, and general intestinal health.

LAB play an important role in the taste and quality of kimchi, and many strains associated with kimchi show antimicrobial activity in addition to other useful properties.

The over-ripening (or over-souring) of kimchi is the most serious defect in kimchi, and is mainly due to the overproduction of lactic acid

by some strains of LAB.

The best way to overcome the problem of excessive acid production is to control the growth of LAB without harming the quality of the end product.

The major objective of the reported work was to study the functional properties of LAB from kimchi

實驗架構

strains isolated from kimchi

Physiological propertiesDetermination of functional properties for selection of

strains

16S rDNA sequencing bystrains isolated from kimchi

6 tests 8 tests

1、 Strains * 16S rDNA sequencing2、 Physiological properties * Carbohydrate fermentation *Hydrolysis of Arginine *Dextran production *Presence of haem-dependent catalase activity *Determination of lactic acid enantiomers

produced *Detection of meso-diaminopimelic acid (m-DAP) 3、 Determination of functional properties for

selection of strains *Response to simulated stomach duodenum-

passage (SSDP) *Determination of cell surface hydrophobicity *Adhesion properties to human cell lines (HT29

cell cultures) *In vitro adherence assay. *Agar diffusion method for antibiotic resistance *Detection of biogenic amine production *Cholesterol-related activities *Antimicrobial activit

Materials & MethodsV.S

Results

Strains

Strains Lactic acid bacterial strains were isolated

from home-made and commercial kimchi using MRS agar for plating.

pH of MRS-broth to 4.1 - 4.2 after 24 h at 30 °C.

These strains were identified by 16S rDNA sequencing using BLAST.

Strains The srains were isolated at levels between

106 and 107/ml from kimchi samples.

Fermentation ability in simulated kimchi juice was checked, and pH values between 3.4 and 3.9 were reached within 24 h.

L. sakei : 11 strain L. plantarum: 1 strain

Physiological properties

Carbohydrate fermentation In MRS borth fermentation broth without

glucose but with chlorophenol red (0.04 g/l) as pH indicator.

The individual sugars were prepared as 2.5% (m/v) solutions and filter sterilized

using a 0.45 m filterμ 25 l of sugar solution was addedto 100 μ μl

Hydrolysis of Arginine The experiment for the production of

ammonia from arginine was carried according to Harrigan and McCance (1976).

MRS broth without ammonium citrate was used as growth medium

Dextran production The strains were streaked onto MRS agar

supplemented with 10% sucrose (Junsei, Japan)

After 24 - 48 h incubation at 30 °C, colonies were investigated for dextran (slime)

production Non-producers showed no change, and had

normal shape and color.

Presence of haem-dependent catalase activity

Cells were grown aerobically at 30 °C in MRS broth containing 5 g glucose/l (Sigma, USA) supplemented with 30 μg haematin (Sigma,

Switzerland).

After 24 h, catalase activity was determined by the presence of effervescence when adding 0.6% H202

Determination of lactic acid enantiomers produced

Strains were grown at 30 °C for 18-24 h in MRS broth with and without sodium acetate.

Lactic acid enantiomers were determined using an enzymatic kit

Based on the oxidation of D(-)-lactate and L(+)-lactate to pyruvate in the presence of NAD+

Detection of meso-diaminopimelic acid (m-DAP)

Presence of meso-DAP in the bacterial cell wall was determined using thin-layer chromatography on cellulose plates

The test described byTamang et al. (2000).

Detection of meso-diaminopimelic acid (m-DAP)

有些菌種的 L-Lysine 會被 meso-Diaminopimelic acid 所置換。 比如大腸桿菌：Escherichia coli 。

Determination of functional properties for selection of strains

Response to simulated stomach duodenum-passage (SSDP)

This assay was designed to represent a simplified and standardized test system

Giving predictive values for the assumed survival of LAB in the human stomach and duodenum under simulated physiological

conditions

The test described by Haberer et al. (2002).

Response to simulated stomach duodenum-passage (SSDP)

Determination of cell surface hydrophobicity

The test for bacterial adhesion to hydrocarbon was adopted to screen lactobacilli for cell surface hydrophobicity (Doyle and Rosenberg, 1995)

For hydrophobicity testing, bacteria were grown in MRS broth at 37 °C for 18 h.

Determination of cell surface hydrophobicity

Cells from 5 ml of culture were collected by centrifugation (9500×g) at 4 °C for 6 min.

The cells were washed twice with PBS.

One ml of this suspension was used to determine the OD580……(1)

Determination of cell surface hydrophobicity

In duplicate assessments, a further 1.5 ml of this suspension was added to an equal volume

of n-hexadecane (Sigma, USA) and thoroughly mixed for 2 min using a vortex.

The phases were allowed to separate at room temperature for 30 min, after which one ml of the watery phase was removed and the OD580

was determined……(2)

Determination of cell surface hydrophobicity

The OD580 of duplicate assessments was meaned and used to calculate the degree of hydrophobicity.

Percentage hydrophobicity was calculated as follows:

OD580 reading 1- OD580reading 2 OD580 reading 1 ×100=% hydrophobicity

Adhesion properties to human cell lines (HT29 cell cultures)

Mucus secreting HT29 cells were routinely grown in RPMI 1640 medium supplemented with 10% bovine calf serum (BCS),and 100 μg

streptomycin and 100 U penicillin per mlat 37 °C in a 5% CO2 atmosphere.

For adhesion assays, HT29 monolayers were prepared in 24-well Costar® tissue plates (Corning Incorporated, USA) according to Schillinger et al. (2005).

Adhesion properties to human cell lines (HT29 cell cultures)

Cells were inoculated at a concentration of 5.4×105 cells per well to obtain confluence and cultured for 21 days prior to the adhesion assay.

In vitro adherence assay The probiotic strain Lactobacillus

rhamnosus GG was used as control

Determination of cell surface hydrophobicity and adhesion properties to human cell lines

Agar diffusion method for antibiotic resistance

The selected 12 strains were screened for their susceptibility to 8 antimicrobial agents

Lactobacillus plantarum 299v as a relevant probiotic control strain

Agar diffusion method for antibiotic resistance

In the MRS and then these cultures were diluted with the same fresh medium to a density 105 CFU/spot for 18 hours at 37 °C.

Plates were incubated in air at 37 °C for 48 h and were examined for growth.

Agar diffusion method for antibiotic resistance

Minimum inhibitory concentration (MIC) was considered as the lowest concentration of the antimicrobial that inhibits bacterial growth

Antibiotic resistance

Detection of biogenic amine production

All strains were subcultured 5 to 10 times in MRS broth containing 0.1% of each precursor amino acid in order to promote the enzyme induction.

Sigma, USA *L-Tyrosine disodium salt *L-Histidine monohydrochloride monohydrate *L-Ornithine monohydrochloride *L-Lysine monohydrochloride

Detection of biogenic amine production

As a positive control, Enterococcus faecalis ATCC 29212 was used, and a positive reaction with tyrosine was detected.

All selected strains were negative in this test

Cholesterol-related activities All selected strains showed deconjugation

ability of bile salts, as was indicated by the BSH test on agar plates

Cultures were screened for bile salt hydrolase (BSH) activity by spotting 10 l of μa culture grown in MRS broth onto BSH screening medium

Cholesterol-related activities MRS agar plates supplemented with 0.5%

(w/v) sodium salt of TDCA (taurodeoxycholic acid) and 0.37 g CaCl2/l

Plates were incubated anaerobically in an anaerobic jar (Difco, USA) at 37 °C .

After which BSH activity was semi-quantified by measuring the diameter of precipitation zones..

Cholesterol assimilation in vitro was determined by growing cells

In MRS broth supplemented with 0.5% w/v TDCA and 0.1 g of water soluble cholesterol/l at 37 °C .

Following incubation, bacterial cells were harvested by centrifugation (10,000 rpm,

for 5 min) and the supernatant and un-inoculated control MRS broth

Cholesterol lowering effects

Antimicrobial activity Using the paper disc method, 100 l culture μ

of each test strain were spread on LB agar plate by sterile swab.

Sterile 6 mm discs (Whatman,England) were placed on the agar and 40 l of μcultures of selected LAB strains were inoculated onto the disc.

Antimicrobial activity After incubation for 12 h at 37 °C, the

diameter of each clear zone was determined.

Hydrogen peroxide production was determined according to Eschenbach et al. (1989).

Presumptive bacteriocin formation was determined according to Choi et al. (2000).

Antimicrobial activity

Discussion

Carbohydrate fermentation The 9 L(+) lactic acid producing strains of L.

sakei hydrolyzed arginine, fermented a wider array of sugars, and showed diversity in their ability to ferment amygdalin, cellobiose, mannitol, trehalose, gentiobiose and salicin

The 2 DL lactic acid could only ferment 6 (galactose, glucose, fructose, mannose, N-acetyl-glucosamine, salicin) out of 49 sugars

survive conditions mimicking of the gastrointestinal tract

Thus, strains such as L. plantarum NR74 L. sakei NR24 and NR65, showing survival rates of N10%.

May be expected to reach the “target” site, the small intestines, in sufficiently high numbers.

adhesion capabilities L. sakei strains showed different adhesion

capabilities

strains did not adhere to HT-29 cells at a high rate

L. sakei NR29 and NR65 showed relatively high adhesion level of around 24%, ＞ that of L. rhamnosus GG (7.3% in this test),

Inhibit the food-borne pathogens Listeria innocua KACC 3586 Listeria monocytogenes KCCM 40307 Bacillus cereus ATCC 08715 Staphylococcus aureus KCCM 11335 Escherichia coli ATCC 25922 Salmonella Typhimurium ATCC 14028 Pseudomonas aeruginosa ATCC 27853 Weissella koreensis KACC 11853

antimicrobial properties The antimicrobial properties, for some strains at least, are due not only to organic

acid production, but also to H2O2 and bacteriocin formation.

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