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The Annals of the University Dunarea de Jos of Galati

Fascicle VI Food Technology, 2003



80
THE STUDY OF ANTIFUNGAL ACTION EXPRESSED
BY SOME BACTERIAL SELECTED STRAINS
Margareta Lcrmioara ZARA*, Nastasia BELC**, Gabriela BAHRIM*, Aida VASILE*
* Galati Dunarea de Jos University, 111, Domneasca St., 800201-Galati, Romania
** Food Bio-resources Institute, Bucharest
Abstract
Romanian food industry passed into a new developing stage and had to align itself to the demands
The aim of this study was to evidentiate the antifungal activity of some bacteria upon the moulds
that are frequently presented in the air of bread-making factories and could contaminate the bread.
Key words: lactic acid bacteria, propionic bacteria, antifungal action
Rsum
L'objectif de cette tude a t la mettre en evidence de l'activit antifongique de quelque souches
de bactrie slectionne sur les moisissures qui sont frquemment actuel dans l'air du fabrique de
pain S.C. Dobrogea S.A. et pourrait contaminer le pain.
Rezumat
Cercetrile ntreprinse n aceast lucrare au urmrit punerea n eviden a efectului antifungic al
unor specii de bacterii selecionate din medii naturale asupra unor tulpini de mucegaiuri prezente
n microflora aerului din spaiile de producie ale S.C. Dobrogea S.A.
1. Introduction
Romanian food industry passed into a new
developing stage and had to align itself to the
demands of the present microbiological world
standards and specifications. Therefore it has
to produce healthy food, to use minimal
processing of raw materials and to improve the
shelf life of food by replacing the synthetic
preservatives with natural substances. In these
conditions, it was necessary to intensify the
research in order to find new ways to inhibit
the activity of spoilage microorganisms that
accidentally contaminate food during
processing or storage.
2. Materials and methods
Twenty bacteria pure cultures were isolated
and tested for their antifungal activity. Using
identification keys there were identified as
strains of Lactobacillus, Streptococcus,
Lactococcus and Propionibacterium.
As indicator microorganisms, there were used
mould pure cultures that were isolated from the
air in the production area of a bread-making
factory (S.C. Dobrogea S.A. Constanta).
Based on their morphological characteristics
there were identified as Penicillium expansum,
Aspergillus oryzae, Aspergillus repens and
Trichothecium roseum.
The following culture media were used to
isolate and cultivate the bacteria:
MRS (Man, Rogosa and Sharpe) and
Rogosa agar for lactic acid bacteria
TYG (Tripticase - Yest Extract Glucose)
for propionic acid bacteria.
The malt extract agar (MEA) was used to
isolate and cultivate the moulds. The same
medium was also used to test the antifungal
activity expressed by the selected bacteria.
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The antifungal activity was successively tested
using the method of radial diffusion through
gels using sterile paper disks impregnated with
bacteria cultures or wells made into the agar
medium that were filled with the bacterial
suspension.
Another way of testing the fungal activity was
to simultaneously inoculate by the pour plate
technique one of the tested bacteria and one of
the indicator moulds (incorporation method).
3. Results and discussion
Applying the classical techniques of isolation
pure cultures of moulds whose incidence is
quite high in the bread-making industry were
obtained.
Analysing the macroscopically and
microscopically properties of the pure culture
it was possible to identify them as Aspergillus
oryzae, Aspergillus repens, Penicillium
expansum and Trichothecium roseum.
On the other hand, 20 pure cultures of bacteria
(table 1) identified as species of the following
genera: Lactobacillus, Streptococcus, and
Propionibacterium were isolated from natural
substrates (borsch, pickles, sauerkraut, milk,
dairy products).
To reveal the bacteria able to produce
antifungal compounds was necessary in the
first stage. This was done using the radial
diffusion through gels and deposing filter
paper disks impregnated with bacterial culture
liquid on the surface of the malt extract agar,
medium which was inoculated with mould
spores by the pour plate technique.
After 5 days of incubation at 2528C the
results presented in table 2 were obtained.
Table 1. The morphological characteristics of the isolated bacteria cultures
Culture
Code Species
Isolation
source
Morphological characters
1 Lactobacillus sp. Sauerkraut Short rods, arranged in angles
2 Lactobacillus plantarum Sauerkraut Short rods with rounded edges, arranged in chains
3 Lactobacillus delbrueckii
ssp. Delbrueckii
Borsch Thin rods forming chains
4 Lactobacillus delbrueckii
ssp. Delbrueckii
Borsch Thin rods arranged in angles
5 Streptococcus sp. Milk Oval cells associated as diplococci and streptococci
6 Lactobacillus sp.
Lactococcus sp.
Curdled milk Short rods arranged in chains
Streptococci forming long chains
7 Streptococcus.
thermophilus
Yoghurt Cells as coccus, arranged as diplococci or short chains
8 Lactobacillus sp. Borsch Short rods with straight edges
9 Lactobacillus delbrueckii
ssp. bulgaricus
Yoghurt Straight or curved rods forming chains
10 Streptococcus.
thermophilus
Yoghurt Oval cells occurring as diplococci and streptococci
11 Lactobacillus plantarum Pickles Short rods with straight edges forming chains
12 Lactobacillus sp. Borsch Short rods with straight heads
13 Lactobacillus sp. Pickles Short rods with straight heads
14 Lactobacillus plantarum Pickles Short rods with straight heads, single
15 Streptococcus sp. Curdled milk Cells as coccus, arranged as diplococci or short chains
16 Lactobacillus delbrueckii
ssp. bulgaricus
Yoghurt Long straight or curved rods forming chains
17 Propionibacterium sp. Schwaitzer Gram positive rods, catalase-positive
18 Lactobacillus sp. Pickles Thin rods, no chains
19 Lactobacillus sp. Curdled milk Straight or curved rods
20 Lactobacillus sp. Pickles Short rods having straight edges, arranged in chains
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Table 2. The potential of the isolated lactic and propionic bacteria to inhibit the growth of the indicator moulds
Indicator moulds Bacterial
culture code
Aspergillus glaucus Aspergillus oryzae Penicillium expansum Trichotecium roseum
1 - - - - - - - - - - - -
2 - - - - + - - - - -
3 + - - + + - - - - - - -
4 + + + + + + +++ +++
5 - - - + - - + - - + + -
6 - - - + - - + - - - - -
7 - - - - - - - - - - - -
8 - - + + - - - - - - -
9 - - - - - - - - - - -
10 - - - + + - + + - + - -
11 - - - - - - - - - - -
12 + + - + + + + - - + - -
13 - - - - - - - - - - -
14 - - - - - - - - - - -
15 - - - - - - - - - - - -
16 - - - - - - - - - - - -
17 - - - - - - - - - - - -
18 - - - - - - - - - - - -
19 - - - - - - - - - - - -
20 - - - - - - - - - - - -
Legend: + + + high inhibition degree; + + - medium inhibition degree; + - - weak inhibition degree; - - - no inhibition
Analysing the data presented in table 2 it could
be concluded that 13 of the tested bacteria
have antifungal activity, their inhibitory effect
varying according to the nature of the tested
mould. The highest inhibitory degree is
manifested by the culture no. 4.
To confirm the results, another method
conceived to test the antifungal activity was
also applied. The results obtained are showed
in table 3.
Table 3. The effect of some lactic and propionic bacteria upon the growth of some moulds
Test bacteria Indicator microorganism
P. expansum A. repens A. oryzae Trichothecium roseum
Name Code
Diameter of the growth inhibition zone, mm
Lactobacillus delbrueckii
ssp. delbrueckii
3 0 0 0 0
Lactobacillus delbrueckii
ssp. delbrueckii
4 18 20 18 11
Streptococcus sp. 5 0 0 0 0
Lactobacillus sp. 8 0 0 0 0
Streptococcus. thermophilus 10 0 0 0 0
Lactobacillus sp. 12 20 14 20 13
Propionibacterium sp. 17 0 15 0 0
Lactobacillus sp. 20 0 0 0 0
Analysing the data presented in table 3 the
following conclusions could be drawn:
Only 3 of the tested cultures manifest
antifungal activity (cultures coded 4, 12
and 17);
Culture 17 presents an antifungal effect
only upon Aspergillus repens;
Culture 12 has a much powerful are effect
on P. expansum and A. oryzae than on A.
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repens, as compared to culture no. 4 that
inhibits more the A. repens strain;
Trichothecium roseum is less sensitive to
the action of antifungal compounds
released by the tested bacteria as compared
to the moulds of the genera Penicillium and
Aspergillus.
The species of the genus Aspergillus
manifest different sensitivities to the action
of a certain antifungal compound.
To confirm the results, the antifungal activity
of culture no. 4, 12 and 17, using inocula, three
days older than previous was tested again. The
well method was used and the same cultivation
conditions were maintained (table 4).
Analysing the data presented in table 4 and
comparing them to that from table 3 one could
conclude that:
The conclusions of the first experiment are
confirmed;
The antifungal activity is dependent on the
culture age.
The antifungal activity of the cultures number
4 and 12 was then tested using the pour plate
method. Each culture was simultaneously
inoculated with one of the indicator moulds.
Each bacteria inoculum was obtained from a 6-
days aged culture. The concentration of the
spores in the fungal inocula was constant. The
developed colonies were counted both in the
witness plates and in the test plates after
incubating them during 3 days at 28C (table
5).
One could notice that the culture number 4 has
a larger antifungal spectrum than the culture
number 12. Thus, the first culture succeeds to
inhibit all the moulds taken as indicators, while
the culture number 12 is able to inhibit only
the growth of Aspergillus repens.
Due to the fact that the indicator moulds are
frequently occurring on the bakery products, it
is recommended to study the activity of the
culture number 4 (Lactobacillus delbrueckii
ssp. delbrueckii) as potential antifungal agent
for such products.
Table 4. The antifungal effect of some lactic and propionic bacteria
Tested strains Indicator microorganisms
P. expansum A. repens A. oryzae Trichothecium roseum
Name Code
Diameter of the inhibition zone, mm
Lactobacillus delbrueckii
ssp. delbrueckii
4 13 18 14 13
Lactobacillus sp. 12 16 12 17 9
Propionibacterium sp. 17 0 10 0 0
Table 5. The antifungal activity of two lactic bacteria strains using the incorporation method
Indicator microorganism
P. expansum A. repens A. oryzae
Culture
code
W T I M T I M T I
4 215 0 100 400 0 100 250 0 100
12 215 0 100 400 116 71 250 0 100
W number of colonies in the witness plate (without bacterial culture)
T number of colonies in the test plate
I inhibition level, %
4. Conclusions
20 pure cultures of bacteria were isolated
from the microbiota of a variety of lactic
and propionic fermented food products.
They were morphological characterised and
identified as being species of the genera
Lactobacillus, Lactococcus, Streptococcus
and Propionibacterium.
Four strains of moulds were isolated from
the air of a bread-making factory in order to
be used as indicator microorganisms for
testing the antifungal activity of some lactic
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and propionic bacteria. The moulds were
identified as Aspergillus repens Aspergillus
oryzae, Penicillium expansum and
Tricotecium roseum.
Applying qualitative and quantitative
methods it was studied the potential of
some lactic and propionic bacterial cultures
to produce antifungal compounds. The
researches put into evidence the fact the
variation of growth inhibition level with the
nature of the indicator mould, the fungal
spore concentration and the age of the
tested bacteria.
The culture coded no. 4 (Lactobacillus
delbrueckii ssp. delbrueckii) presents the
best antifungal activity having in view the
inhibition potential and activity spectrum.
Therefore, this culture is recommended to
be used as antifungal agent.
5. References
Al-Zoreky, N., Sandine, W.E. (1991). Lactococcus
genus. A selective and differential agar medium,
J.Food Sci., 56, 6, 1286-1288.
Bahrim, G. (1999). Microbiologie tehnic. Evrika
Publishing House, Braila.
Bibek, R. (1996). Fundamental Food Microbiology,
CRC Press.
Bouix, M. and Leveau, J.Y. (1993). Controle
microbiologique. In: SCRIBAN, R. et al. eds.
Biotechnologie (4
th
edition, entirement
refondue, actualise et augumente), Paris.
Bourgeois, C.M. and Leveau J.Y. (1991). Les
techniques danalyse et de contrle
microbiologique (tome 3), Lavoisier, Paris.
Dan, V. et al. (1999). Memorator mucegaiuri,
Evrika Publishing House Braila.