Impact factor-2.

812

By
Poorna Chandra Rao K
INTRODUCTION
 Food-Borne pathogenic bacteria-endanger human
health, severe losses to food stuffs

 Chemical additives-inhibit the growth of bacteria but

it is harmful to human health besides environment.

 Lactic acid bacteria-Inhibitory compounds, against

spoilage and pathogenic bacteria

 Bacteriocin-safe bio-preservative, sensitivity to

proteolytic enzymes

 Lactobacillus sakei, found in diverse habitats.

 It is used as a starter cultures in dry sausage
production

 Important characteristic of bacteriocins produced

by L.sakei is their activity against Listeria species

 Promising biological food preservatives especially

in meat products

 In the present study a novel bacteriocin termed

sakacin LSJ618 was isolated from traditional
Chinese fermented radish

 Further reports the characteristics and mode of

action of sakacin
 MATERIALS AND METHODS
 Strains, Media and Culture conditions
 24 different types of traditional Chinese
fermented foods were obtained for the study.

 L.sakei LSJ618 and other indicator strains were

isolated from traditional Chinese fermented
radish

 MRS and LB medium

 Stored at -700c in MRS or LB supplemented with

DMSO
ISOLATION OF BACTERIOCIN PRODUCING
STRAINS
Traditional Chinese fermented radish in 50ml
sterilized water

Serial dilution

Plated on MRS agar with 0.01%bromocresol purple

incubation
Colonies that change the color of the medium from purple to
yellow

Restreaked onto MRS plates

Bacteriocin activity by agar well diffusion test

IDENTIFICATION OF THE
STRAINS
 Initially by phenotypical and physiological tests

 Further by sequence analysis of 16S rDNA.

 DNA isolation with UNIQ-10 Column bacterial

Genomic DNA Isolation Kit

 16S rDNA was amplified by PCR with primer P1 (5’-

AGAGTTTGATCCTGGCTCAG-3’), and P2 (5’-
TACGGTTACCTTGTTACGACTT-3’)using a PCR
amplification kit

 The PCR products were sequenced and homologies

were examined by comparing DNA databases.
PREPARATION OF CRUDE
BACTERIOCIN
 Cell free supernatant by centrifugation(9000 g
for 15 min) and filter sterilization (0.45 mm
pore-size membrane).

 It was then dialyzed against double distilled

(dd) H2O for 6 h at 4oC

 The solution was then lyophilized

 The resulting supernatant powder (SP) was used

as the sample for further analysis
ASSAY OF BACTERIOCIN ACTIVITY
AND ANTIBACTERIAL SPECTRUM
 Agar well diffusion test (Ennahar et al.,2000)
using Micrococcus tetragenus as the indicator
strain

 Diameter of the inhibition zone around the

wells, and expressed as arbitrary units (AU) per
ml

 One AU was defined as the reciprocal of the

highest serial two-fold dilution producing
distinct inhibition of the indicator lawn
(Todorov & Dicks, 2005)
PRELIMINARY CHARACTERIZATION
OF SAKACIN LSJ618
 Supernatant powder(SP) was used in the agar well
diffusion test

 Thermostability test by heating the SP at

60oC,80oC,100oC and 121oC and then placed in an ice bath
for 10min

 Residual bacteriocin activity was then determined

 pH stability test by adjusting the SP to pH 2,4,6,8,10

and12 with 1.0M NAOH & 1.0M HCL

 After 6hr of incubation at 37oC, samples were readjusted

to pH 6.0 and tested for residual bacteriocin activity
 Sensitivity to hydrolytic enzymes was
determined by incubating the SP for 6hr with
6mg/ml of α-chymotrypsin, protease K, trypsin,
lipase, papainase and pepsin at 37oC

 After incubation samples were heated for 10min

at 100oC to inactivate the enzyme

 Residual bacteriocin activity was determined

 Effect of chemical reagents was determined by

incubating for 4hr with 1% Tween 20, Tween 80,
urea, SDS or EDTA at37oC

 Residual bacteriocin activity was examined

 LSJ618
 5% seed culture of M.tetragenus was cultured and
when the growth reached late logirthmic phase,
320 AU/mL of sakacin LSJ618 was added

 OD600 and viable cell count was determined once

every hour.

CELL GROWTH, pH FLUCTUATUIONS AND

PRODUCTION OF SAKACIN LSJ618
 Strain LSJ618 was inoculated in MRS broth and

incubated

 OD600,pH and the antibacterial activity against

M.tetragenus was determined for every 2hr .
PARTIAL PURIFICATION AND MOLECULAR
WEIGHT OF SAKACIN LSJ618
Cell free supernatant

Precipitated with 80% ammonium sulfate saturation at 4oC

Proteins isolated by centrifugation (9000 g for 45 min at 4oC) and

resuspended in ddH2O

Dialyzed for 6 h at 4oC & lyophilized

lyophilized sample was run in a 2.6 cm x 100 cm Sephadex G-25

column

Active Fractions with bacteriocin activity were then analyzed by

Tricine-SDS-PAGE.
 Later the gel was Stained with Coomassie
brilliant blue for molecular weight
determination.

 Also antimicrobial assay on the gel was

performed by washing the gel with sterile water
and overlaying with M.tetragenus seeded
nutrient soft agar.

 Culturing at 37oC to determine the inhibitory

effect(Gao et.al., 2010)
RESULTS AND DISCUSSION
ISOLATION AND IDENTIFICATION OF
THE BACTERIOCIN-PRODUCING STRAIN
 Strain LSJ618 was isolated from a particular kind
of Chinese fermented radish

 It was a rod-shaped, Gram-positive, catalase-

negative bacterium with white circular colonies
on MRS plates.

 Grew in MRS broth from 4oC to 41oC, but not at

45oC and grew in 7.5% NaCl, but not 10% NaCl.

 To further characterize the strain, fermentable

carbohydrates were examined and 16S rDNA
sequences were analyzed.
Phylogenetic tree derived from the 16S rDNA sequence of the L. sakei
strain LSJ618 showed 99% similarity to that of L. sakei BMG168 .Hence
it was indicated that LSJ618 was a L. sakei strain.
EFFECTS OF DIFFERENT TREATMENTS ON THE
ANTIBACTERIAL ACTIVITY OF SAKACIN LSJ618
ANTIBACTERIAL SPECTRUM OF
SAKACIN LSJ618
PARTIAL PURIFICATION AND MOLECULAR
WEIGHT OF SAKACIN LSJ618
PRODUCTION OF SAKACIN LSJ618

Production of sakacin LSJ618 during the growth of L. sakei LSJ618 in

MRS broth at 37 C. Activity of bacteriocin (◦), optical density at
600 nm (□) and changes in pH (▲).
MODE OF ACTION OF SAKACIN
LSJ618

The effect of sakacin LSJ618 on the growth of M. tetragenus. Data shown are
optical density at 600 nm without (▲) and with (Δ) sakacin LSJ618 and viable
cell counts (CFU/ml) without (●) and with (○) sakacin LSJ618.
 CONCLUSION
 In the present study, a new bacteriocin-producing strain,
L. sakei LSJ618, was isolated from the traditional Chinese
fermented radish.

 Sakacin LSJ618 was stable to changes in environmental

factors.

 Sakacin LSJ618, ~5.2 kDa lipoprotein produced by L. sakei

LSJ618, inhibited the growth of food-spoiling bacteria and
food-borne pathogens

 Furthermore, its mode of action was determined to be

bactericidal.

 These properties indicate that sakacin LSJ618 has

potential utility as a biopreservative in the food industry.
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 Ennahar, S., Sashihara, T., Sonomoto, K., & Ishizaki, A. (2000).
Class IIa bacteriocins: biosynthesis, structure and activity. FEMS
Microbiology Reviews, 24, 85e106.
 Todorov, S. D., & Dicks, L. M. T. (2005). Lactobacillus
plantarum isolated from molasses produces bacteriocins active
against Gram-negative bacteria. Enzyme and Microbial
Technology, 36(2-3), 318e326.
 Schillinger, U., & Lücke, F. K. (1989). Antibacterial activity of
Lactobacillus sake isolated from meat. Applied and
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 Prasad, S., Morris, P. C., Hansen, R., Meaden, P. G., & Austin,
B. (2005). A novel bacteriocin-like substance (BLIS) from a
pathogenic strain of Vibrio harveyi. Microbiology-Sgm, 151,
3051e3058.
 Gao, Y., Li, D., Sheng, Y., & Liu, X. (2011). Mode of action of
sakacin C2 against Escherichia coli. Food Control, 22, 657e661.
Thank you