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Table 1.
Lactic acid bacteria (LAB) studied.
Name of the strain
Strain code (abbreviation)
Origin
Lactobacillus plantarum
VTT E-78076
(E76)
Beer
L. plantarum
VTT E-79098T
(E98)
Pickled cabbage
Pediococcus pentosaceus
VTT E-90390
(E390)
Barley kernel
P. pentosaceus
VTT E-981081
(E1081)
Oat
L. amylovorus
VTT E-95576T
(E576)
Silage
L. amylovorus
VTT E-981145
(E1145)
Starch slurry
L. acidophilus
VTT E-96276T
(E276)
Human isolate
0.0
1.2
0
48
96
Time (hrs)
1.0
10
% M
0
-10
-20
-30
-40
-50
-60
-70
-80
-90
-100
-110
-120
-130
0
10
5
20
30
40
15
25
35
50
45
60
55
70
65
80
75
90
85
95
105
0119-003
0119-007
0119-009
0119-013
0119-015
0119-018
Time (hrs)
SCREENING OF THE ANTIFUNGAL
EFFECT OF LACTIC ACID BACTERIA
AGAINST TOXIGENIC
PENICILLIUM
AND
ASPERGILLUS
STRAINS
L. VANNE, T. KLEEMOLA, A. HAIKARA
VTT BIOTECHNOLOGY
P.O.Box 1500, FIN-02044 VTT, +
358 9 455 2103, liisa.vanne@vtt.fi
MATERIALS AND METHODS
The mould strains selected for
this study were 15 identified
Penicillium verrucosum and four
Aspergillus ochraceus isolated
mainly from wheat and barley.
The inhibition of mould growth
was screened with seven lactic acid bacteria (LAB), partly
based on
earlier studies of their antifungal effects (1). A summary of
the LAB used in this study is
presented in Table 1.
AIM
In this study, the possible antifungal effects of lactic
acid bacteria
against toxigenic Penicillium
and Aspergillus strains were
screened in
vitro. The work is a part of the EU project “Preven-
tion of ochratoxin A in
cereals”(QLK1-CT-1999-00433).
A second screening with the
four most potentially
antifun-
gal LAB was carried out with
autoclaved barley kernels as a growth substrate in
order to simu-
late in vivo conditions. The
effect of LAB culture supernatants
on the growth of fungi was monitored by indirect
impedimetry
(BacTrac 4100, Sy-Lab, Austria). The principle of the test
method is
presented in Figure 2. The CO2
production of the fungi was
monitored for four days at 25 °C and the detection
time of
the set conductance threshold value was used as a measure of
fungal growth. The delay in CO2
production was considered to
result from the inhibitory
effects of the LAB studied.
ACKNOWLEDGMENTS
We thank M.Sc. Hanna-Leena Alakomi for her valuable
advice in the implementa-
tion of the impedance technique and Ms. Pirjo Tähtinen and
Ms. Tarja Nordenstedt
for excellent technical assistance.
REFERENCES
1. Haikara, A. and
Mattila-Sandholm, T. 1994. Procedure for treatment of seed
material to be germinated.
Patent WO9416053 (Pat.FI 94875, 1995).
2. Skyttä, E. and
Mattila-Sandholm, T. 1991. A quantitative method for assessing
bacteriocins and other food
antimicrobials by automated turbidometry.
J. Microbiol. Methods 14, 77-88.
3. Niku-Paavola, M-L., Laitila,
A., Mattila-Sandholm, T. and Haikara, A., 1999.
New types of antimicrobial
compounds produced by Lactobacillus
plantarum.
J. Appl. Microbiol. 86, 29-35.
RESULTS
The antifungal effect measured by automated
turbidometry
varied between 20 and 55 %. The set inhibition level
of 40 % was
achieved in 22 of 104 combinations of LAB and
P. verrucosum.
The results with the three
most effective LAB strains, Lacto-
bacillus plantarum
E76, L. plantarum E98 and L. amylovorus
E576 are presented in Figure 3.
The inhibition of A. ochraceus
by LAB was found to be more
than 40% in 11 out of 26 combina-
tions (Figure 4.). The inhibition caused by LAB is a
combined
effect of lactic acid and antimicrobial substances produced
by the bacteria (3).
Figure 1.
Kinetic turbidity measure-
ment by Bioscreen®
analyser. Spores
of P. verrucosum (1 x 104
pmy) are
in- cubated
together with LAB culture
filtrates in a total volume of 300 ml
of growth
medium (PD broth).
1.
Control
2.
Control with MRS and lactic acid
3-4. L.
plantarum E76 and E98
5.
L. amylovorus
E576
Figure 2. Test conditions of
indirect
impedimetry. Ten autoclaved barley
kernels are incubated with
fungal
spores and LAB culture filtrates in a
cryovial. The detection time of
CO2
production by fungi is used as
the pa-
rameter to estimate growth inhibition.
Figure 3.
Effect of three LAB culture supernatants on the growth of
P. verrucosum
strains in liquid culture.
Fungal strains
1.
VTT D-98495
2.
VTT D-00807
3.
VTT D-00754
4.
VTT D-00753
5.
VTT D-00749
6.
VTT D-00748
7.
VTT D-00798
8.
VTT D-00799
9.
VTT D-00831
10. VTT D-00823
11. VTT D-00826
12. VTT D-00750
13. VTT D-00800
14. VTT D-98494
15. VTT D-00752
Figure 4. Effect
of four LAB culture
supernatants on the growth of A.
ochraceus
strains in liquid culture.
Figure 5. Effect of three LAB
culture
supernatants on the CO2
production of
A. ochraceus VTT D-00808 in auto-
claved barley.
1.
Control, 2. Control with
MRS broth,
3. P.
pentosaceus E390, 4.
Control
with MRS broth and lactic acid, 5.
L.
plantarum
E98, 6.
L. plantarum E76
1
2
3
4
5
6
1
2
3-4
5
CONCLUSIONS AND FUTURE PLANS
The results achieved in this
study show clearly that the growth
of toxigenic storage fungi can be restricted by LAB
in vitro.
Further studies with irradiated barley as
growth substrate are
going to show the potential of the technique in barley
storage
and processing.
All the four LAB strains studied
using the indirect impedimetric
method were able to delay or
even inhibit the growth of
A. ochraceus and P.
verrucosum
during the 90-hour monitoring period. The most effective
strains,
L. plantarum E76 and L. amylovorus
E576, were able to inhibit
the growth of all 14 fungal strains tested. In Figure
5, an example
of the results is shown.
The preliminary screening of
the antifungal effect of LAB was
carried out by automated
turbidometry (2). Fungal spores (appr.
104
spores per test well) and cell-free culture filtrates of LAB
grown in MRS broth were
incubated in a total volume of 300 ml
in an integrated
incubator-photometer instrument (Bioscreen®,
Labsystems, Finland). The
turbidity was measured periodically
by kinetic vertical
photometry for 96 hours at 25 °C. Growth
inhibition of moulds caused by
LAB was calculated by compar-
ing the area under the growth curve with the area
in control
samples. An example of the primary measurement data is
shown in
Figure 1.
Screw cap
with safety
valve
Barley
kernels in
a cryovial
KOH-
resistant electrode
plug
1 ml
0.2 KOH
0
10
20
30
40
50
60
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
E76
E98
E576
E1145
E576
0
10
20
30
40
50
60
E76
E98
VTT D-96650
VTT D-00785
VTT D-00808
VTT D-00826
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