Gaining more from pilot-plant studies: an example for VTEC
in fermented semi-dried sausages
Tina Beck Hansen1, Annemarie Gunvig2, Flemming Hansen2 and Søren Aabo1
National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark
2 DMRI, Danish Technological Institute, Maglegaardsvej 2, DK-4000 Roskilde, Denmark
INTRODUCTION & OBJECTIVE
CONCLUSIONS
A pilot-plant study often test behaviour of cocktails of strains
giving the opportunity to cover more biological variability in
the study. However, a possible strain variation will be masked,
as the most tolerant strain will be the one recovered and
enumerated.
In the present work, VTEC with different indigenous antibiotic
resistant markers were used to disclose the biological
variability between strains during production of fermented
semi-dried sausages.
FIG. 1. Survival of VTEC during production of semidried sausages
VTEC O111
6
6
0 ppm NaNO2
5
6
0 ppm NaNO2
5
4
4
3
3
3
2
2
2
1
1
1
0
0
0
0
7
14
21
28
0
7
14
21
28
6
100 ppm NaNO2
5
100 ppm NaNO2
5
3
3
3
2
2
2
1
1
1
0
0
7
14
21
28
200 ppm NaNO2
7
14
21
28
200 ppm NaNO2
5
3
3
3
2
2
2
1
1
1
0
0
0
21
28
6
5
5
4
4
3
3
2
0
7
14
21
28
2
4.5 (dotted line)
5.0 (full line)
pH
1
14
21
28
6 % (full line)
12 % (dotted line)
WPS
1
0
0
2
4
6
8
10
12
14
16
0
2
4
6
8
10
12
14
16
Incubation at 16°C, days
7
14
21
Log-reduction
28
4.5-5
4-4.5
3.5-4
3-3.5
200 ppm NaNO2
5
4
14
7
6
VTEC O157
FIG. 3. Confidence in log-reductions obtained during
production of semi-dried sausages
0
4
7
7
6
4
0
7
0
0
6
5
8
100 ppm NaNO2
5
4
VTEC O111
0
0
4
6
VTEC O26
8
6
4
0
0 ppm NaNO2
5
4
6
VTEC O157
6 % WPS
log10CFU/mL
5 % WPS
FIG. 2. Survival of VTEC in broth model systems
200
150
100
50
0
7
14
21
28
Starter culture F-1 Bactoferm, production day
4
4.5
5
5.5
% WPS
0
6
EXAMPLE:
4.5 % WPS
75 ppm NaNO2
Standard deviation
200
0.6-0.8
0.4-0.6
0.2-0.4
0-0.2
0.8
0.7
0.6
150
100
0.5
0.4
0.3
50
0.2
0.1
0
2
3
4
5
6
Log-reduction
4
4.5
5
5.5
NaNO2 (ppm)
Concentration of VTEC in product (log10CFU/g)
VTEC O26
4 % WPS
 Strain variability increased with addition of NaNO2 to the
sausage batter (Fig. 1)
 Strain variability occurred earlier for 4 and 6 compared to
5 % water phase salt (WPS) in the sausage batter (Fig. 1)
 Strain variability was unaffected by type of starter used in
the sausage
 Strain variability in broth was inconsistent with results
obtained in semi-dried sausages (Fig. 2)
 Low NaNO2 combined with low WPS gave the highest logreduction as well as the highest confidence (Fig. 3)
Predicted NORM (4.1,0.48)
1
0
6
% WPS
MATERIALS & METHODS
RESULTS
A cocktail of equal volumes of VTEC O26, O111 and O157, were prepared from overnight
cultures grown in BHI at 37°C for 24 h. Three independent pilot-plant studies were
performed where the cocktail was added during chopping of the meat. Survival during
production of fermented, semi-dried sausages were investigated. Fermentations were
performed at 24°C for 48 h, drying at 16°C until 35 % weight loss. Agar substrates with
relevant antibiotics were developed to recover each of the strains separately (Tab. 1). The
studies covered recipes with varying water phase salt (WPS) contents (approx. 4, 5 and
6%), sodium nitrite levels (0, 100 and 200 ppm) and starter cultures (F-1 Bactoferm and
FSC 111 Bactoferm from Chr. Hansens A/S) used in the batter.
Pooled results from three pilot-plant studies
(Fig. 1) showed that independent on WPS
content and starter culture, the strain
variability was lowest when no NaNO2 was
used. In sausages, with added NaNO2 (100
and 200 ppm), containing 4 % or 6 % WPS,
strain variability typically peaked after 10 d,
whereas in sausages with 5 % WPS, strain
variability was highest after 24 d. This
change in strain variability during production
was primarily a result of low survival of
O111, as compared to O26 and O157.
These observations were inconsistent with
the survival observed in broths containing
levels of lactate (Fig. 2 – pH) and NaCl (Fig.
2 – WPS) comparable to the sausages.
TAB. 1. Counts (log10CFU/mL) of overnight VTEC cultures (BHI, 37°C)
VTEC
strain
BHI
agar
SMaC
CT-SMaC
MaC
MaC + 64 µg/L
streptomycin
MaC + 16 µg/L
neomycin
O26
9.43
9.34
Growth
(red)
9.48
No
growth
9.23
O111
9.61
9.62
No
Growth
9.66
9.49
No
growth
O157
9.51
9.57
9.43
(colourless)
9.48
No
growth
No
growth
Modelling the survival of VTEC obtained
after 24 d showed the highest confidence in
log-reduction estimates for the sausages
prepared from batter containing 4 to 4.5 %
WPS and 0 to 50 ppm NaNO2 (Fig. 3).