Transcript Lactobacillus plantarum

Safety aspects of application of
Lactic Acid Bacteria
SVETOSLAV TODOROV
Federal University of Viçosa
Viçosa, MG, Brazil
Lactic acid bacteria
Early definition of LAB as a group, based on the ability to
ferment and coagulate milk, included coliform bacteria with
the lactic. The description of Lactobacillus organism by
Beijerinck in 1901 as Gram-positive bacteria separated the
coliforms from LAB. According to Orla Jensen in 1919 “the true
LAB form a natural group of Gram-positive non-motile, nonspore forming, rod- and coccus-shaped organisms that ferment
carbohydrates and higher alcohols to form chiefly lactic acid.”
Lactic acid
bacteria
Rods
Cocci
Lactobacillus
Lactococcus, Enterococcus,
Carnobacterium
Streptococcus, Leuconostoc,
Weisella, Pediococcus
Streptococcus gallolyticus subsp.
macedonicus
Desemzia, Isobacilum,
Paralactobacillus, Tetragenococcus,
Trichococcus, Oenococcus,
Melissococcus
Lactic acid bacteria
medicine
food and feed industries
USES
Lactic acid bacteria
chemistry
Lactic acid bacteria
FUNCTIONAL INGREDIENTS
medicine
food and feed industries
chemistry
USES
Lactic acid bacteria
probiotics
FUNCTIONAL INGREDIENTS
enzymes
vitamins
starter cultures
dairy
non-dairy
Antimicrobial agents
biopreservatives
medical sector
sweeteners
exopolysaccharides
medicine
food and feed industries
chemistry
USES
Lactic acid bacteria
probiotics
FUNCTIONAL INGREDIENTS
enzymes
vitamins
starter cultures
dairy
non-dairy
Antimicrobial agents
biopreservatives
medical sector
sweeteners
exopolysaccharides
Antimicrobial agents produced by LAB
organic acids
carbone dioxide
hydrogen peroxide
diacetyl
bacteriocins
low molecular weight substances
All act as biopreservatives in food, with records dating
back to approximately 6000 B.C.
(Pederson, 1971; De Vuyst and Vandamme, 1994).
Bacteriocins & Probiotics
www.scopus.com
Bacteriocins & Probiotics
www.scopus.com
Bacteriocins & Probiotics
www.scopus.com
Bacteriocins & Probiotics
www.scopus.com
What are bacteriocins?
Classical definition
Bacteriocins produced by LAB are polypeptides synthesized in
ribosomes, that exhibit bactericidal or bacteriostatic effect
against genetically closely related bacteria
Klaenhammer, 1988, De Vuyst & Vandamme, 1994
Bacteriocins: developing innate immunity for food
Paul D. Cotter, Colin Hill & R. Paul Ross
Nature Reviews Microbiology 3, 777-788 (October 2005)
Bacteriocins of LAB
Mechanisms of action
Cell wall
Cell membrane
Bacteriocins: developing innate immunity for food
Paul D. Cotter, Colin Hill & R. Paul Ross
Nature Reviews Microbiology 3, 777-788 (October 2005)
Spectrum of activity
Bacteriocin
Closely related LAB
Important Food or
Human Pathogens
Other LAB,
included
LAB from
same ecological
niche
Unusual activity: Gram-negative, viruses, TB, Yeast and Fungi
A
B
Lactobacillus plantarum ST8Sh
Shpek
M
1
2
3
4
5
300 bp
100 bp
Lactobacillus plantarum
Agarose gel showing DNA fragments obtained after
PCR with species-specific primers.
Lines 1 and 2: strain ST8SH,
lines 3 and 4: Lactobacillus plantarum LMG 13556,
line 5: No DNA and
line M: O’GeneRulertm 100 bp DNA Ladder
(Fermentas).
Lactobacillus plantarum ST8Sh
Plantaricin S
Nisin
Plantaricin NC8
Pediocin PA-1
Plantaricin W
Lactobacillus plantarum ST8Sh
Plantaricin S
Nisin
Plantaricin NC8
Pediocin PA-1
Plantaricin W
Test microorganism
Bacteriocin activity
Enterococcus faecalis
8/8*
Enterococcus faecium
6/9
Enterococcus mundtii
1/1
Escherichia coli
0/2
Klebsiella pneumoniae
1/3
Lactobacillus paracasei
2/2
Lactobacillus paraplantarum
0/2
Lactobacillus pentosus
2/2
Lactobacillus plantarum
5/7
Lactobacillus sakei
3/4
Lactobacillus salivarius
2/2
Lactobacillus delbruekii
0/2
Lactobacillus fermentum
0/2
Lactobacillus curvatus
2/5
Lactococcus lactis subsp. lactis
3/3
Leuconostoc mesenteroides
0/3
Listeria innocua
4/4
Listeria monocytogenes
103/108
Listeria ivanovii subsp. ivanovii
1/1
Pediococcus acidilactici
0/3
Staphylococcus aureus
0/5
Spectrum of activity
recorded for
bacteriocin ST8Sh
Growth of
Lactobacillus plantarum ST8SH
in MRS broth (-▲-),
changes in pH (-●-) and
production of bacteriocin
expressed in AU/ml (histogram
bars)
at 370C (A),
300C (B) and
250C (C).
Effect of different
concentrations of
bacteriocin ST8SH on
(A) Listeria monocytogenes
ScottA,
(B) Lactobacillus sakei ATCC
15521 and
(C) Enterococcus faecalis ATCC
19433
recorded at 3h, 9h and 18h
presented as % of lysis of
test microorganisms.
Effect of bacteriocin ST8SH
on exponentially growing
(a) Listeria monocytogenes
ScottA,
(b) Lactobacillus sakei ATCC
15521 and
(c) Enterococcus faecalis
ATCC 19433.
(-♦-) without added
bacteriocin; (-▲-) with
added 10% (v/v)
bacteriocin ST8Sh
Growth of a mixed culture of
Lactobacillus plantarum ST8SH and
Listeria monocytoegnes ScottA (--) and
Listeria monocytogenes ScottA (--).
Changes in pH is shown by --.
Production of bacteriocin ST8SH is indicated
by the histogram
The dose makes the poison,
a principle of toxicology, was first
expressed by Paracelsus.
It means that a substance can
produce the harmful effect
associated with its toxic properties
only if it reaches a susceptible
biological system within the body
in a high enough concentration
(dose).
Paracelsus
(Philippus Aureolus Theophrastus Bombastus von Hohenheim, 11
November or 17 December 1493 – 24 September 1541)
was a German-Swiss Renaissance physician, botanist, alchemist,
astrologer, and general occultist
Toxicity of bacteriocin ST8Sh
Cell-free supernatant of
Lactobacillus plantarum ST8Sh
Partial purification of the
bacteriocin ST8Sh
Semi-purified fractions (60% iso-propanol
and 80% iso-propanol) of bacteriocin ST8SH
were tested on Huh7.5 cells for their cytotoxicity profile
Toxicity of bacteriocin ST8Sh
Cell-free supernatant of
Lactobacillus plantarum ST8Sh
Partial purification of the
bacteriocin ST8Sh
Semi-purified fractions (60% iso-propanol
and 80% iso-propanol) of bacteriocin ST8SH
were tested on Huh7.5 cells for their cytotoxicity profile
At concentration of 25 µg/ml
60% fraction demonstrated
to be highly cytotoxic, reducing
the cell viability by
approximately 80%, but not with
lower tested concentrations of
the bacteriocin ST8Sh
Regarding the 80% iso-propanol
Fraction of bacteriocin ST8Sh,
cell viability
was not reduced in
both tested concentrations
(25 µg/ml and 5 µg/ml)
Toxicity of bacteriocin ST8Sh
» Toxicological studies showed that nisin intake does
not cause toxic effects to the human body with an
estimated lethal dose (LD50) of 6950 mg/kg, which
is similar to that of salt, when administered orally
(Jozala, Andrade, Arauz, Pessoa Junior & VessoniPenna, 2007).
» In general, some authors have associated LD50 of
bacteriocins with digestive enzymes capable of
rapidly inactivating these substances such as trypsin
and chymotrypsin produced in the pancreas
(Deegan, Cotter, Hill, & Ross, 2006; Motta,
Cannavan, Tsai & Brandelli, 2007; Vaucher et al.,
2011).
Beneficial properties of Lactobacillus plantarum ST8Sh
Survival in GIT conditions
Beneficial properties of Lactobacillus plantarum ST8Sh
Survival in GIT conditions
Growth/survival in presence
of low pH and oxbile
Beneficial properties of Lactobacillus plantarum ST8Sh
Survival in GIT conditions
Aggregation propertis
Growth/survival in presence
of low pH and oxbile
Beneficial properties of Lactobacillus plantarum ST8Sh
Survival in GIT conditions
Aggregation propertis
Growth/survival in presence
of low pH and oxbile
Interaction with antibiotics
and commercial drugs
Beneficial properties of Lactobacillus plantarum ST8Sh
Survival in GIT conditions
Aggregation propertis
Hydrophobicity
Growth/survival in presence
of low pH and oxbile
Interaction with antibiotics
and commercial drugs
Beneficial properties of Lactobacillus plantarum ST8Sh
Survival in GIT conditions
Aggregation propertis
Growth/survival in presence
of low pH and oxbile
Interaction with antibiotics
and commercial drugs
Hydrophobicity
β-galactosidase
Beneficial properties of Lactobacillus plantarum ST8Sh
Survival in GIT conditions
Aggregation propertis
Growth/survival in presence
of low pH and oxbile
Interaction with antibiotics
and commercial drugs
Hydrophobicity
β-galactosidase
Adherence to cell lines
Caco2 & HT29
Beneficial properties of Lactobacillus plantarum ST8Sh
Survival in GIT conditions
Aggregation propertis
Growth/survival in presence
of low pH and oxbile
Interaction with antibiotics
and commercial drugs
Hydrophobicity
β-galactosidase
Adherence to cell lines
Caco2 & HT29
Cholesterol reduction
Beneficial properties of Lactobacillus plantarum ST8Sh
Some genes
Mub
EF2662-cbp
Mab
EF1249-fbp
EF-tu
EF2380-maz
prgB
(surface protein)
Beneficial properties of Lactobacillus plantarum ST8Sh
Some genes
Mub
EF2662-cbp
Mab
EF1249-fbp
EF-tu
EF2380-maz
prgB
(surface protein)
Safety of Lactobacillus plantarum ST8Sh
Lactobacillus plantarum ST8Sh
Safety of Lactobacillus plantarum ST8Sh
Antibiotic resistance
Production of biogenic amines
Virulence factors
Horizontal gene transfer
Safety of Lactobacillus plantarum ST8Sh
Antibiotic resistance
Production of biogenic amines
Virulence factors
Horizontal gene transfer
ddl
E. faecalis
aac(6′)-Ii
E. faecium
mur-2ed
mur-2
E. durans
E. hirae
vanC1
vanC2
(D-ala-D-ser ligase) (D-ala-D-ser ligase)
vanC1
vanC2/C3
ermA
ermB1
ermC
ermB2
vanB
tetK
tetL
tetM
tetO
tetS
aac(6′)-Ie-aph
(2″)-Ia
aph(3′)-IIIa
ant(6)-Ia
catA (PIP501)
vatE
ccf
cob
bcrB
bcrD
bcrR
cpd
sprE
efaA
ace
esp
cylA
hdc2
odc
aph(2″)-Ib
(phosphotransferase)
hdc1
tdc
fsrC
fsrA
gelE
asa1
fsrB
int
hyl
int-Tn
vanA
ant(4′)-Ia
aph(2″)-Id
aph(2″)-Ic
(adenylyltransferase)(phosphotransferase)(phosphotransferase)
Safety of Lactobacillus plantarum ST8Sh
Antibiotic resistance
Production of biogenic amines
Virulence factors
Horizontal gene transfer
ddl
E. faecalis
aac(6′)-Ii
E. faecium
mur-2ed
mur-2
E. durans
E. hirae
vanC1
vanC2
(D-ala-D-ser ligase) (D-ala-D-ser ligase)
vanC1
vanC2/C3
ermA
ermB1
ermC
ermB2
vanB
tetK
tetL
tetM
tetO
tetS
aac(6′)-Ie-aph
(2″)-Ia
aph(3′)-IIIa
ant(6)-Ia
catA (PIP501)
vatE
ccf
cob
bcrB
bcrD
bcrR
cpd
sprE
efaA
ace
esp
cylA
hdc2
odc
aph(2″)-Ib
(phosphotransferase)
hdc1
tdc
fsrC
fsrA
gelE
asa1
fsrB
int
hyl
int-Tn
vanA
ant(4′)-Ia
aph(2″)-Id
aph(2″)-Ic
(adenylyltransferase)(phosphotransferase)(phosphotransferase)
Are we going to open the Pandora’s box???
Lucius Annaeus Seneca (often known simply as
Seneca; ca. 4 BC – AD 65) was a Roman Stoic
philosopher, statesman, dramatist, and in one work
humorist, of the Silver Age of Latin literature. He was
tutor and later advisor to emperor Nero.
I know that I know
nothing, but you even
don’t know this.
[email protected]
Svetoslav D. Todorov