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
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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"
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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.
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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.
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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.
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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
16S rDNA sequencing by
strains isolated from kimchi
Physiological properties
6 tests
Determination of functional
properties for selection of strains
8 tests
1、Strains
* 16S rDNA sequencing
2、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 & Methods
V.S
Results
Strains
Strains
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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
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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
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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 μm filter
25 μl of sugar solution was addedto 100 μl
Hydrolysis of Arginine
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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
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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
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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
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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 (mDAP)
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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)
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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
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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
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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
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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
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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)
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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)
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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
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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
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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
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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
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Minimum inhibitory concentration (MIC)
was considered as the lowest concentration
of the antimicrobial that inhibits bacterial
growth
Antibiotic resistance
Detection of biogenic amine production
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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
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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
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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 μl of
a culture grown in MRS broth onto BSH
screening medium
Cholesterol-related activities
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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
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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 uninoculated control MRS broth
Cholesterol lowering effects
Antimicrobial activity
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Using the paper disc method, 100 μl culture
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 μl of
cultures of selected LAB strains were
inoculated onto the disc.
Antimicrobial activity
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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
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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, Nacetyl-glucosamine, salicin) out of 49 sugars
survive conditions mimicking of
the gastrointestinal tract
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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
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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
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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
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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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