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. The Annals of the University Dunarea de Jos of Galati Fascicle VI Food Technology, 2003
81 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 The Annals of the University Dunarea de Jos of Galati Fascicle VI Food Technology, 2003
82 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. The Annals of the University Dunarea de Jos of Galati Fascicle VI Food Technology, 2003
83 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 The Annals of the University Dunarea de Jos of Galati Fascicle VI Food Technology, 2003
84 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.