Transcript BACTERIOCINS - Synthetic Biology Conferences

Lactic acid bacteria for
nutraceuticals and
added value products
production
Enrica PESSIONE
Department of life Sciences and Systems Biology.
University of Torino- Italy
London, August 18th 2016
In spite of the long established use of LAB , their
biotechnological
potential
nutraceutical industry is still:
• Underexplored
• Underexploited
for
the
food
and
LACTIC ACID BACTERIA
Biocontrol
agents
Probiotics
Food
starters
Cell
factories
Our researches on LAB include
Selenoproteins
Neuroactive
compounds
(GABA, PEA)
PROBIOTICS
Technological stability
during lyophilization
Bioactive peptides
from food proteins
EPS
Bacteriocins
Nutraceutical production
PROTEOMICS
Reconstructing
metabolic pathways
Evaluating stress
resistance
Optimizing the
technological operative
conditions
Detection
of
enzymes
involved
generation of desired metabolites:
• Proteases
• Glycosyltransferases
• Amino acid decarboxylases
• Selenocysteine lyase
in
Detection of proteins controlling stress:
• Acid stress
• Oxidative stress
• Starvation
• Osmotic stress
• Competition stress ( co-cultures)
• Heat/cold stress
SUBPROTEOMES
Probiotic LAB releasing seleno
proteins
Selenium plays an important role in:
• Antioxidant activity
• tyroid function
• healthy immune response
• viral infections
• regulation of inflammatory pathways
• protection against some forms of cancers
• ageing
Selenium in nature
TAVOLA PERIODICA degli ELEMENTI
Solo 30 dei 90 elementi sono essenziali (in rosso) per gli organismi viventi; hanno
numero atomico basso; quelli più abbondanti sono H, O, N, C.
Essi sono tra gli elementi più leggeri; formano uno, due, tre, quattro legami
chimici; costituiscono il 96% della materia vivente.
Gli elementi in tracce (in giallo) sono essenziali alla vita perché necessari per il
funzionamento degli enzimi (catalizzatori delle reazioni chimiche).
Selenium chemical forms
• Selenium can be present in different chemical forms:
• Selenite
• Selenate
• Elemental selenium
• Selenomethionine
• Selenocysteine
Some form are toxic or not bioavailable for humans
SELENIUM-FIXING LAB
some Lactobacillus strains ( L.reuteri Lb2 BM) are able
to fix Selenium depending on the oxidation state of Se:
• when it is added to the medium as selenate only a very
low amount of Se was intracellularly detected
• conversely the highest Se concentration in the
biomass
was
obtained
(Na2SeO3= 4.38 mg/L)
with
sodium
selenite
SELENIUM-FIXING LAB
• the excess of sodium selenite not used for insertion into
proteins is reduced to elemental selenium Se(0).
• This is a non-toxic form of Se that produces deposits at
surface level giving a red color to the biomass (Lamberti
et al. 2011).
A
B
C
E
F
G
D
H
SEM analyses reveal nanoparticles
on the cell surface which confer a
red color to the biomass for the
reduction of Se(IV) to Se (0)
COMPARATIVE L. reuteri Lb2 BM PROTEOME
(grown with and without SELENIUM)
Soluble Proteome
C Lamberti et al. Proteomic characterization of
a
selenium‐metabolizing
probiotic
Lactobacillus reuteri Lb2 BM for nutraceutical
applications. Proteomics 11 (11), 2212-2221
SELENIUM-FIXING LAB
Differently from what happens in yeasts that fix
Se as selenomethionine, L.reuteri Lb2 BM
seem
to
selenocysteine
incorporate
selenium
as
ICP mass experiments: Several proteins contain selenocysteines: two of them are secreted
COMPARATIVE EXTRACELLULAR
PROTEOME
• Lactobacillus reuteri Lb2BM is able to fix Se in 2 extracellular
proteins
(phosphoketolase and GAP dehydrogenase) as
SeCys.
• In the near future Se-enriched lactobacilli could be used as food
supplements to supply Sec-containing proteins.
LAB PRODUCING
NEUROMODULATING COMPOUNDS
GABA
Gamma amino butyric acid
•
•
•
•
•
•
Neurotransmission
Immune modulation
Anxiety control
Insomnia
Depression
Hypotensive
Gamma amino butyric acid is produced as the
result of the peculiar energy metabolism of LAB
mainly based on lactic fermentation. ( No heme, no
respiration)
Amino acid decarboxylations coupled with antiport
systems allow to increase the proton gradient
across the membrane together with cytoplasm
basification
AMINOACID
DECARBOXYILASE
AMINOACID/AMINE
ANTIPORTER
AMINOACIDO
AMINE
+
PROTONIC GRADIENT
H+
AMINOACID
AMINE
CO2
+
- - + + +
-
+ +
+
PMF
It is possible to enhance GABA production by acting on the
LAB growth conditions:
• Adding glutamate, the precursor amino acid, or glutamine.
Open
question:
glutamate
cost:
Corynebacterium
glutamycum co-culture or spent medium? Which wastes?
Mazzoli et al, Amino Acids, 2010
It is possible to enhance GABA production by acting on the
LAB growth conditions:
• Maintaining acidification. Optimization by evaluating the
stress proteome
Mazzoli et al, Amino Acids, 2010
HDC at pH 8.0
catalytically unactive
HDC at pH 4.8
catalytically active
It is possible to enhance GABA production by acting on the
LAB growth conditions:
• Maintaining a high chloride content (transcriptional activator of GAD
gene) Optimization by evaluating the stress proteome
• Adding arginine to supply an additional NH3 donor for transaminations
to direct all glutamate or glutamine to GABA
GABA-TEA
GABA-RICE
YOGURT
FERMENTED
SOYBEAN
BIOACTIVE PEPTIDES
BIOACTIVE PEPTIDES
•
•
•
•
Antioxidant
Metal chelating
Antimicrobial
Immunomodulating
• Antithrombotic
• Antihypertensive
• Cholesterol lowering
• Opioid
• Anti-opiod
Infection
Inflammation
Ageing
Cardiovascular
diseases
Anxiety
Depression
Mood
BIOACTIVE PEPTIDES
3 – 20 aa
LAB PROTEOLYTIC SYSTEM
The proteolytic system of three
LAB strains was evaluated towards
GLUTEN and CASEIN
Gluten bioactive peptides
blank
L. RHAMNOSUS 17D10 late logarithmic
No signals for
gluten-derived
peptides in
mass
spectrometry
for the three
strains
analyzed
L. RHAMNOSUS 17D10 late stationary
L. HELVETICUS 4D5 late logarithmic
L. HELVETICUS 4D5 late stationary
L. LACTIS MG1363 late logarithmic
L. LACTIS MG1363late stationary
CASEIN hydrolysis L. LACTIS MG1363
Higher amounts of
peptides in logarithmic
phase
Blank
LOG
Fast proteolytic system
STATIONARY
CASEICIN C
(Casein αs1)
SDIPNPIGSENSEK
Antimicrobial peptide
CASEIN hydrolysis L. RHAMNOSUS
17D10
Blank
Higher amounts of
peptides in
stationary phase
High hydrolytic
power of this
strain towards
caseins
LOG
STATIONARY
Antimicrobial peptides by L. rhamnosus 17D10
ISRACIDIN
(Caseina αs1)
RPKHPIKHQGLPQEVLNENLLRF
(Hayes et al., 2007)
CASOCIDIN
(Caseina αs2)
KTKLTEEEKNRLNFLKKISQRYQKFALPQYLKTVYQHQK
LOW-COST SUBSTRATES
Milk whey proteins
BACTERIOCINS
BACTERIOCINS
• Antibacterial compounds generally produced under quorum sensing
control
• Active a very low concentration ( nanomolar)
• Small peptides or proteins
• Secreted or surface-bound
• LAB are good producers: nisin, lactacine, enterocine, reutericine,
sakacine, salivaricine, mutacine, gassericine
BACTERIOCINS
•
Promising tools for infection control as antibiotic substitutes
(urgent issue!)
•
During about 50 years use of nisin no resistance spread.
•
Useful in prolonging shelf-life protecting food from spoilage
bacteria, thus reducing the use of preservatives, high salt,
high sugar, cold chain.
•
Useful
to
control
monocytogenes)
food-borne
infections
(Listeria
CLASS I: inhibition of peptidoglican synthesis
CLASSE II: pore
forming at
cytoplasm
membrane
level,
dissipation of
the proton
gradient
CLASS III: cell-wall
hydrolysis
P.D. Cotter et al. 2005
BACTERIOCINS
Cellular localization
Max production
Strain
Spectrum of activity
Growth
O2 effect
CFS
effect
-O2
+O2
Interfering
Stability
Proteinase
molecule
K
identification
CS
Lb. plantarum 37A
List. monocyt and S. aureus
LS (48 h)
+
-
+
-
Resistant
lactic acid
E. faecium G12
List. monocytogenes
LE (18 h)
+
++
+
+
Sensitive
Enterocin A
E. faecium G6
List. monocytogenes
LE (18 h)
+
++
+
+
Sensitive
Enterocin A
L. lactis 5
List. monocyt and S. aureus
LE (18 h)
+
-
+
+
Sensitive
Nisin A
L. lactis 7
List. monocyt and S. aureus
LE (18 h)
+
-
+
+
Sensitive
Nisin A
L. lactis 8A
List. monocyt and S. aureus
ES (18 h)
+
-
+
+
Sensitive
Nisin A
L. lactis 8B
List. monocyt and S. aureus
ES (18 h)
+
-
+
+
Sensitive
Nisin A
L. lactis 15
List. monocyt and S. aureus
ES (18 h)
+
-
+
+
Sensitive
Nisin A
L. lactis cremoris EL3
List. monocyt and S. aureus
EE (6 h)
+
-
+
+
Sensitive
Nisin A
BACTERIOCINS: overlapping gel test
BACTERIOCINS
• Some limits concern their peptide/protein nature: they are easily degraded by
proteases both in the food matrix ( during cheese ripening) and in vivo (
gastrointenstinal system)
• A promising strategy is to immobilize them into the food packaging, after suitable
enrichement and purification.
• The advantage of food packaging-immobilized bacteriocins, « active packaging»
results in :
a) Limited and controlled diffusion into the food matrix, limited degradation
b) Release in a concentration gradient
c) Protection from inactivation
BACTERIOCINS: challenges
Growing LAB on a lowcost substrate
Stimulate production
(oxigen stress, co-coltures
in membrane fermenter,
bacteria hydrolysates)
Optimizing recovery
(Recover also surfacebound by shaving)
Film-bacteriocin
compatibility
(Enhanced diffusion into
polymers)
Improve performances
conservative modifications to:
• Extend the antibacterial spectrum
• Enhance protease resistance
EXOPOLYSACCHARIDES
EXOPOLYSACCHARIDES
food industry
•
•
•
Thickeners
Texture improvement
Stabilizers for low-fat products
human health
• Prebiotics (FOS= low molecular mass EPS)
• Immunomodulating
• Cholesterol lowering
biotechnological industry
• Chromatographic media
EPS
LAB CAN
SYNTHESIZE homoEPS DIRECTLY FROM SUCROSE BY MEANS OF
GLYCANSUCRASE THAT HYDROLYZES SUCROSE AND USE THE GLYCOSYL OR
FRUCTOSYL MOIETY FOR POLYMERIZATION
Enhancers of homoexopolysaccharide production:
• Sucrose
• Temperature: the highest the growth temperature
the lowest EPS production. Preliminary proteomic
results on L.plantarum Q8.23 reveal induction of
stress proteins during active growth ( 37°) but not
under EPS production conditions ( 30°).
EPS production by L.plantarum Q8.23 grown at 30°or 37°
30ºC
30ºC
37ºC
37ºC
MRS ctrl
A
B
C
D
E
0,824
0,853
0,801
0,84
0,762
0,827
0,847
0,807
0,85
0,79
0,8255
0,85
0,804
0,845
0,776
Factor de dilución
mg/L EPS
Promedio Desviación estándar
mg/L EPS producidos por L. plantarum
45,97
16
287,30
240,91
65,60
139,44
48,63
25
194,52
43,63
40
109,08
114,65
7,88
13,18
48,09
40
120,22
40,59
40
101,47
101,47
0,00
Quantitative determination of EPS. There was
an
inverse
relationship
between
EPS
production and growth temperature. The EPS
production of L. plantarum Q8.23 was significantly
higher at 30ºC (139.44 mg/L) than 37ºC (7.88
mg/L).
• The results of our researches demonstrate that LAB potential is
much higher than expected and that it is possible to exploit them
for nutraceuticals and added value chemical production.
• It is important to continue to study LAB
proteomes to enhance our knowledge on
possible future applications in both industrial
and medical fields.
THANK YOU!