THE IN VITRO ANTIFUNGAL ACTIVITY OF LACTOBACILLUS SPP. AGAINST ASPERGILLUS OCHRACEUS GROWTH AND ITS OCHRATOXIN A PRODUCTION

the genus Lactobacillus on growth and ochratoxin A production of the microscopic filamentous fungus Aspergillus ochraceus (SLO-B-245). Nine species of the genus Lactobacillus were used in this study, namely L. bifermentatus. L. perolens, L. reuteri, L. paracasei, L. paraplantarum, L. plantarum, L. salivarius, L. fructivorans and L. pentosus . All strains were isolated from dairy products and their identification was performed by MALDI TOF MS (Bruker Daltonics, Germany, Maldi Biotyper). The antifungal activity was carried out using the overlay technique. Solid culture media inoculated with bacterial and fungal inocula were cultivated under aerobic conditions at 37 ºC for 8 days. Diameters (Ø mm) of the grown fungal colonies were measured every two days. To determine the inhibition of ochratoxin A production, the thin-layer chromatography method (TLC) was used. L. plantarum and L. paraplantarum were able to inhibit the growth of A. ochraceus (SLO-B-245) completely (100% inhibition), while L. fructivorans appeared to be the least effective (10.73%). Interestingly, the latter one was able to completely inhibit or partially suppress ochratoxin A production. We hope that our results can contribute to the search for harmless substances for the biological control of fungi in food. Lactobacilli and similar organisms could be very promising alternatives for food preservation.


INTRODUCTION
Contamination of food, feed and other agricultural commodities by microscopic filamentous fungi leads to significant economic losses worldwide. Among Aspergillus species are one of the most common microscopic fungi causing degradation of stored food and feed. In addition, some species of the genus Aspergillus pose a major security risk due to their ability to produce mycotoxins (Oliveira et al., 2014). Some of them are toxic and may have carcinogenic, teratogenic or mutagenic properties that can cause various acute or chronic conditions in humans and animals. High incidence of contamination caused by microscopic filamentous fungi and their products -mycotoxins in food and feed, their ability to colonize different substrates and the lack of effectiveness of existing control measures require more effective action to be taken to eliminate these microorganisms . At present, the food industry depends on chemical preservatives (such as benzoic, propionic, and sorbic acids), which can extend the shelf life of foods and control the growth of toxigenic fungi. On the other hand, there is a growing awareness of the health risks of consumers associated with the use of chemical preservatives (Luz et al., 2017). The demand for foods that do not contain these synthetic substances has also been increasing recently. In addition, the use of synthetic preservatives is beginning to be limited due to the resistance development in microscopic filamentous fungi (Muhialdin et al., 2020). In recent years, there has been a growing interest in research and development of natural antimicrobial agents capable of inhibiting the growth of spoilage fungi in food (Ribes et al., 2018). Replacing synthetic food chemicals by natural compounds promotes production of healthier food products that are protected against microscopic spoilage (Gamba et al., 2015;Hati et al., 2018). One of the substitutions is the use of certain microorganism that must be non-toxic and easy to handle growth manifestations on simple media. Lactic acid bacteria (LAB) represent such an alternative as they are known to be a potential source of various secondary metabolites, such as bacteriocins, organic acids, fatty acids, protein compounds, cyclic dipeptides, hydrogen peroxide and other compounds  ., 2021). So, the aim of this study was to monitor the antifungal effect of selected types of lactobacilli on the growth of Asperillus ochraceus isolated from bread and its ability to produce ochratoxin A.

Antagonistic microorganisms -identification, culture conditions and inoculum preparation
Overall, nine strains of the genus Lactobacillus were used in this study as antagonistic microorganisms, specifically L. bifermentatus. L. perolens, L. reuteri, L. paracasei, L. paraplantarum, L. plantarum. L. salivarius, L. fructivorans and L. pentosus. All strains were obtained previously by isolation from dairy products and their identification was performed by MALDI TOF MS (Bruker Daltonics, Germany, Maldi Biotyper) as described in Hleba et al. (2017). All the strains were cultivated in MRS broth (HiMedia, India) under aerobic conditions at 37 °C for 48 hours in order to obtain bacterial suspensions for inoculums preparation. The inoculums containing 10 6 CFU/ml were prepared by diluting the lactobacilli suspensions in sterile phosphate saline and adjusting the density to 0.5 McFarland units by densitometer.

Fungal strain -identification, growth condition and inoculum preparation
Aspergillus ochraceus (SLO-B-245) was used to determine the inhibitory effect of lactobacilli. This strain was previously isolated from bread samples and identified as A. ochraceus ITS: MH517572 (Císarová et al., 2020). Prior to fungal inoculum preparation, A. ochraceus (SLO-B-245) was grown on Sabourad Dextrose Agar (SDA) (HiMedia, India) at 37 °C for 5-7 days in the dark. The grown colonies were resuspended in 1 ml of sterile phosphate saline containing Tween 80 and homogenized. Obtained suspension was diluted in sterile phosphate saline to a final concentration 2.5 x 10 6 CFU/ml by adjusting the density to 0.9 McFarland units.

Screening of Lactobacillus spp. antifungal activity in in vitro condition
The antifungal activity of the 9 lactobacilli strains was carried out using the overlay technique as described by Magnusson and Schnürer (2001) with some modifications. The lactobacilli strains were cultivated on MRS agar (HiMedia, This study was aimed to monitor the inhibitory effect of selected species of the genus Lactobacillus on growth and ochratoxin A production of the microscopic filamentous fungus Aspergillus ochraceus (SLO-B-245). Nine species of the genus Lactobacillus were used in this India) in Petri dishes (Ø 60 mm) . The sterile Whatman filter paper (Ø 12 mm) was placed in the middle of the prepared MRS agar plates and the lactobacilli inoculum (100 μl ) was applied on it. Plates were incubated for 72 hours under aerobic conditions at 37 °C, subsequently overlaid with 2 ml of SDA medium and inoculated by 20 μl of A. ochraceus (SLO-B-245) inoculum (applied in the center of the SDA medium) (Fig. 1). Inoculated plates were cultivated under aerobic conditions at 37 ºC for 8 days. The control treatment contained sterile Whatman filter paper without bacterial inoculum. Diameters (Ø mm) of the grown colonies were measured every two days (on days 2, 4, 6, and 8 of cultivation) using a digital caliper. The obtained data was expressed as the percentage growth inhibition (PGI) according to the equation: Percentage growth inhibition (%) = 2 2 100 where rA = radius of the inhibition zone; rB = Petri dish radius.

Statistical evaluation
All experiments were performed in triplicates. The percentage of growth inhibition (PGI%) was calculated using the MS Office Excel 2016 program and the obtained averages of fungal colony treated by lactobacilli strains were evaluated by using the Stagraphics centurion XVI (version 16.1.11) (one-way ANOVA and Tukey HSD 95% multiple range test (p˂0.05).

Antifungal activity of the tested lactobacilli
Microscopic fungi are ubiquitous organisms that can grow almost everywhere, however their presence in goods results into the greatest losses in food industry. In food products they cause not only spoilage but also loss of nutrients, discoloration, and odor change, and, of course, they are able to produce some highly toxic secondary metabolites -mycotoxins (Hassan et al., 2016). Nowadays the consumers concern about the safety of food has increased as well as the need for finding alternatives suitable for food preservation without the use of synthetic preservatives (Luz et al., 2017; Luz et al., 2020). Lactic acid bacteria (LAB) attract much attention as they produce various substances with antifungal properties. LAB are considered as "green preservatives" because of their ability to inhibit the growth of microscopic fungi in food. Organic acids are still considered to be the major metabolites of LABs that dramatically affect fungal growth by inhibiting the mycelial growth. Organic acids in protonated or undissociated form are lipophilic and therefore easily diffuse across the fungal cell membrane and accumulate in the cytoplasm (Sadiq et al., 2019).
In this study, the antifungal effect of nine lactobacilli strains (L. bifermentatus. L. perolens, L. reuteri, L. paracasei, L. paraplantarum, L. plantarum. L. salivarius, L. fructivorans and L. pentosus) was tested against ochratoxigenic fungus A. ochraceus . The results (Tab. 1) showed that all tested lactobacilli possess the ability to inhibit the growth of A. ochraceus (SLO-B-245) when compared to control sets. All studied strains caused significant differences (P ˂ 0.05) on the mycelial growth of A. ochraceus . L. plantarum and L. paraplantarum were the most effective species of the set as they inhibited the growth completely (0.00 ± 0.00 mm; PGI 100%) after cultivation period (8 days (Fig. 2). After 4 days of cultivation, the growth of A. ochraceus was stopped by the action of these two species compared to the control sets with the maximum colony diameter size of 9.  The other tested lactobacilli had a moderate inhibitory effect on the growth of A. ochraceus with a PGI ranging from 38.02% (L. bifermentatus, L. perolens) to 41.50%. Interestingly, the species L. salivarius (5.31±1.12 mm) and L. perolens In our study, the least effective species was L. fructivorans, showing only 10.73% growth inhibition of A. ochraceus (SLO-B-245) at the end ofcultivation time. This strain even stimulated the growth of A. ochraceus (SLO-B-245) until the second day of cultivation compared to the control set (Fig. 3, Fig. 4). Similar results obtained Inglin et al. (2015) when testing 504 lactobacillus isolates including L. fructivorans against different bacteria and fungi strains. They found that L. fructivorans does not exhibit any inhibitory activity against both, bacteria, and fungi, respectively. Contamination of food by microscopic filamentous fungi and their toxins represents a fundamental safety risk for humans and animals (Uzsáková and Hlebová, 2021). Therefore, in our work we also monitored the potential ability of lactobacilli to inhibit ochratoxin A production in in vitro conditions by using the TLC method. Only those colonies of A. ochraceus  were chosen for this assay, which were not completely inhibited by lactobacilli in previous experiment. The results showed that lactobacilli acted differently on the production of ochratoxin A (Tab. 2, Fig. 5). The most effective lactobacilli, inhibiting the production of ochratoxin A completely, showed to be the following species: L. perolens, L. paracasei, L. pentosus and L. fructivorans. L. fructivorans was able to completely suppress the production of ochratoxin A by A. ochraceus despite its inability of fungal growth inhibition. Piotrowska and Zakowska (2005) also observed the ability of five species of the genus Lactobacillus (L. plantarum BS, L. brevis, L. acidophilus CH-5, L. rhamnosus GG and L. sanfranciscensis) to inhibit the production of ochratoxin A. In their study the most effective species was L. rhamnosus GG, which was able to reduce the production of ochratoxin A by 89%. The inhibition of ochratoxin A production using lactobacilli was also monitored by Li et al. (2021). Their results showed that Lactobacillus brevis 8-2B strain significantly inhibited the growth of Aspergillus carbonarius as well as its production of OTA. Ragoubi et al. (2021) found out that the species L. delbrueckii and L. acidophilus were able to reduce not only OTA and aflatoxin B1 production but also zearalenone and deoxynivalenol production by 30 to 57% except. Lactic acid bacteria can remove mycotoxins in two ways, either by adsorption through the cell wall or by mycotoxin molecules degradation resulting into less toxic compounds. From safety point of view, adsorption is considered to be a more effective method as the degradation process can create some even more toxic metabolites than the original mycotoxins. Furthermore, various studies have proven that the complex mycotoxin-the cell wall of BMK shows reduced rate of adhesion to mucous membranes (Vinderola and Ritieni, 2015). In addition to antifungal and antitoxinogenic properties of lactic acid bacteria, which include, for example, the genera Lactobacillus or Bifidobacterium, there are many other beneficial effects possessed by these bacteria, boosting consumers' health (Uzsáková and Hlebová, 2021). Legend: ntnot tested (mycelial growth was completely inhibited by lactobacilli), OAochratoxin A, ** -number of mycotoxins produced isolates, * -number of tested isolates Figure 5 The inhibitory effect of some species of lactobacilli on the production of ochratoxin A by the strain A. ochraceus (SLO-B-245) tested by the TLC method under UV light 366 nm; K abc -Control, L a -L. perolens, L b -L. fructivorans, L c -L. reuteri, L d -L. paracasei, L e -L. pentosus, * -repetitions

CONCLUSION
Results obtained in this study proved the antagonist effect of LAB (lactobacilli isolated from dairy products) against filamentous fungus A. ochraceus (SLO-B-245) as well as the ability to inhibit or suppress its ochratoxin A production. Antifungal activity assay revealed that two species were able to inhibit the growth of A. ochraceus (SLO-B-245) completely (100%) during all days of cultivation (8 days), namely L. plantarum and L. paraplantarum. The least effective species was L. fructivorans possessing no inhibitory effect on the growth of the microscopic fungus compared to the control; however, it was able to completely inhibit the production of ochratoxin A. The production of ochratoxin A was completely inhibited also by the species L. perolens, L. paracasei and L. pentosus. The second and the third species showed at the same time a high rate of growth inhibition of A. ochraceus (SLO-B-245). Our study revealed some lactobacilli strains with promising properties such as antifungal activity and ability to inhibit/suppress mycotoxin production. In conclusion, our results emphasize the importance of research on lactic acid bacteria due to their application potential in food protection and biopreservation as well as biological control agents in food against microscopic filamentous fungi and their mycotoxins.