PCR multi-paramétriques

Growth,
Growth metabolic activity and stress of
acid lactic bacteria during manufacture and ripening
of swiss-type Emmental cheese
by TTGE,
TTGE real time and RT-Q-PCR
Florence Postollec
DST week, Rennes April 21st 2009
Goal & experimental set-up

Evaluate growth, metabolic activity and stress level of 4 targeted lactic
acid bacteria during cheese making process by molecular methods
-
Streptococcus thermophilus ST88 (ST88)
ST88
Propionibacterium freudenreichii ITG P14 (P14)
P14
Lactobacillus helveticus LH56 (LH56)
LH56
Lactobacillus paracasei LC225 (LC225)
LC225
-
Pilot scale Emmental cheese productions (triplicata)
-
PCR-TTGE & RT-Q-PCR-TTGE (16S rRNA)
Q-PCR & RT-Q-PCR (x3)
 Metabolic activity (16S rRNA)
 Stress level (GroL)
Growth of lactic acid bacteria during
manufacture & ripening of Emmental
Sampling steps during cheese manufacture & ripening
ST88
LH56
1,0E+10
Bacterial counts during Emmental cheese manufacture and ripening (CFU/g)
LC225
1,0E+09
P14
1,0E+08
1,0E+07
1,0E+06
1,0E+05
1,0E+04
1,0E+03
1,0E+02
1,0E+01
milk + inoculation 6h press
48ÄC
acidification
37ÄC
storage
12ÄC
24ÄC
21
CC
14
CC
7C
C
EC
C
CT
T2
0
T1
0
T4
T0
La
e
1,0E+00
Lfa
b
CFU/g

Metabolic activity and stress level of
lactic acid bacteria during
manufacture & ripening of Emmental

Dominance & metabolic activity by PCR-TTGE
Lb. plantarum
M LAE LFab
Lb. fermentum
Lb. Brevis
Staph. xylosus
T0
M
T10
T20
ADN ARN ADN ARN
M M
CT
ECC
7CC
14CC
ADN ARN ADN ARN ADN ARN ADN ARN
M
21CC
28CC
CF
ADN ARN ADN ARN ADN ARN
M
Lb. Brevis ou
Staph xylosus
E. Faecium ou
Staph. equorum
?
Lb. helveticus
E. Faecalis
ou Staph. succinus
T4
ADN ARN ADN ARN ADN ARN
?
?
?
Microbacterium
gubbeenense
ou Lb. reuteri
S. thermophilus
?
?
Lb. paracasei
Propio.
freudenreichii
M: Lb. plantarum, Lb. fermentum, Enterococcus faecium, Lb. helveticus, Enterococcus faecalis, Lc. Lactis, S. thermophilus,
Corynebacterium moorparkense, Lb. paracasei, Arthrobacterium nicotianae, Brevibacterium casei, Propionibacterium cyclohexanicum
 LH56, ST88: population & metabolic activity observed along the process
 LC225, P14: metabolic activity mainly observed for the last stages of process
Metabolic activity and stress level of
lactic acid bacteria during
manufacture & ripening of Emmental

Metabolic activity & stress level by RT-Q-PCR
CF
T20
CT
ECC
7CC
14CC
21CC
28CC
A-CT
T0
T4
T10
A-T4
Copy number cDNA/DNAx109
Lae
Lfab
Lae
T0
T0
T4
T4
T10
T20
CT
CT
ECC
7CC
14CC
21CC
28CC
CF
Copy number cDNA/DNAx109
Copy number cDNA/DNAx109
Lfab
Lae
T0
T4
T10
T20
CT
ECC
7CC
14CC
21CC
28CC
CF
4
T4
T10
T2
0
T20
C
T
E CT
C
C
7C
ECC
C
14
7CC
C
C
21
14CC
C
C
28
21CC
C
C
28CC
C
F
CF
10
T
m
p
Lfab
T0
Lae
T
it e
la
P. freudenreichii ITG P14
1E+11
1E+10
1E+09
1E+08
1E+07
1E+06
1E+05
1E+04
1E+03
1E+02
1E+01
1E+00
Bacterial count CFU/g
10
,E+09
10
,E+08
10
,E+07
10
,E+06
10
,E+05
10
,E+04
10
,E+03
10
,E+02
10
,E+01
10
,E+00
Bacterial count CFU/g
1E+12
1E+11
1E+10
1E+09
1E+08
1E+07
1E+06
1E+05
1E+04
1E+03
1E+02
1E+01
1E+00
1,0E+14
1,0E+13
1,0E+12
1,0E+11
1,0E+10
1,0E+09
1,0E+08
1,0E+07
1,0E+06
1,0E+05
1,0E+04
1,0E+03
1,0E+02
1,0E+01
1,0E+00
Bacterial count CFU/g
Specific bacterial counts (UFC/ml)
Q-PCR quantification for 16S rRNA () & GroL()
10
,E+14
expressed
as a LH56
ratio of equivalent
genome
Lb. paracasei
LC225
Lb. helveticus
10
,E+13
S. thermophilus ST88
10
,E+12
9
10
,E+11
copy number cDNA/DNAx10
10
,E+10
Bacterial count CFU/g
Copy number cDNA/DNAx109
-
 Metabolic activity & stress response quantified for both dominant & minor populations
 valuable biomarkers to evaluate cellular activity & improve NSLAB quantification
RT-Q-PCR is a discriminative & accurate tool for direct comparative
measures to further study LAB behaviour during dairy product processes
Thanks for your attention  poster C17  [email protected]