The Lactobacillus strains used were previously characterized by Pascual et al. (2008a ,b and Ruiz et al. (2009) as presenting probiotic properties: colonization, self-aggregation, adherence to epithelial cells and coaggregation with bacterial pathogens. Lactobacillus rhamnosus L60 and L. fermentum L23 are producers of secondary active metabolites, such as organic acids, bacteriocins and, in the case of L. rhamnosus L60, hydrogen peroxide. Bacteriocin production was previously characterized and the substance was purified (Pascual et al., 2008a ,b). The two strains showed a wide spectrum of antimicrobial activity against Gram-positive and Gram-negative bacteria, some being human and animal pathogens. The present study shows the potential of L. rhamnosus L60 and L. fermentum L23 in control of Aspergillus section Flavi growth and AFB1 production in vitro. Biopreservation, the use of microorganisms to preserve food and feed stuffs, has been gaining increasing interest due to consumers' demand for reduced use of chemical preservatives (Prema et al., 2010). As LAB are ‘generally recognized as safe’ organisms (Hoque et al., 2010), they could have useful application in the prevention of fungal contamination in raw materials, food and feed, and in reducing the health hazards associated with mycotoxins.
The present study clearly shows the antifungal effect of L. rhamnosus L60 and L. fermentum L23 on aflatoxigenic fungal isolates. Nevertheless, L. rhamnosus L60 was the most effective strain in inhibiting growth of all Aspergillus section Flavi strains assayed in vitro. Our results agree with those reported by Vanne et al. (2000), who assayed the effects of Lactobacillus casei on growth and aflatoxin production by A. parasiticus. Onilude et al. (2005) demonstrated that Lactobacillus plantarum, L. fermentum, Lactobacillus brevis and Lactococcus spp. have in vitro antifungal effects on aflatoxigenic fungal isolates in similar proportions to those detected in this study. The results obtained in the present study agree with those of other researchers, who assayed Lactobacillus species similar to those used in this study but with other LAB strains in the in vitro growth control of Aspergillus spp. and other fungal strains (Magnusson & Schnürer, 2001; Zara et al., 2003; Kam et al., 2007; Muñoz et al., 2010; Voulgari et al., 2010). The growth rate inhibition by lactobacillus strains on fungal species may be caused by production of secondary metabolites. Lactobacillus rhamnosus L60 and L. fermentum L23 are producers of organic acids, bacteriocins and, in the case of L. rhamnosus L60, hydrogen peroxide (Pascual et al., 2008a ,b; Ruiz et al., 2009). The presence of these substances in culture media could inhibit the fungal development of Aspergillus section Flavi species, as observed in our assays. Lactic and acetic acids are the main products of the fermentation of carbohydrates by LAB. These acids diffuse through the membrane of target organisms in their hydrophobic undissociated form and then reduce cytoplasmic pH, thereby causing loss of viability and cell destruction (Gerez et al., 2009; Dalié et al., 2010). Although there is no clear evidence of the role of protein compounds in the inhibition of mould growth, several authors have reported that some lactic strains produced antifungal metabolites that were sensitive to proteolytic enzymes (Magnusson & Schnürer, 2001; Rouse et al., 2008). On the other hand, the strong inhibitory activity can be attributed to competition between LAB and Aspergillus section Flavi species in batch conditions. However, the observed reduction of the lag phase is probably due to rapid adaptation of fungal strains to the culture medium but LAB may have advantages over fungi as they are simpler organisms with a faster metabolim. Therefore, bacteria can utilize the original substrate earlier to produce more cell biomass, while fungi develop later after nutrient levels are lower. We have clearly demonstrated here the inhibitory effect of growth of Aspergillus section Flavi strains by secondary metabolites of LAB. However, future studies will need to determine the optimal concentration of pure organic acid, bacteriocins and hydrogen peroxide that inhibit fungal growth. When fungal isolates were grown in the presence of Lactobacillus strains, AFB1 accumulation showed the same trend as growth rate, with accumulation of this metabolite reduced significantly (P < 0.05). None of the LAB strains stimulated AFB1 accumulation in any of the fungal strains assayed. On the contrary, toxin production of A. flavus RC2053 and A. flavus RC2055 was totally inhibited by L. fermentum L23. It is likely that the low concentration of AFB1 in the presence of Lactobacillus strains could be due to low mycelial biomass formation. Growth inhibition could directly affect AFB1 production as a result of low synthesis of the enzymes involved. Furthermore, AFB1 is a secondary metabolite that does not occur during primary growth of fungus, so that growth inhibition may reduce its production. In this study we have showed that there could exist a relationship between fungal growth and AFB1 production. In fact, these results showed that minimal yields of toxin coincided with minimal mycelial growth. Tukey's test of the data revealed the influence of L. fermentum L23 and L. rhamnosus L60 on growth parameters (lag phase and growth rate) and AFB1 production. Our results agree with Zinedine et al. (2005), who demonstrated the ability of some strains of LAB to reduce the initial concentration of AFB1 in MRS broth. Similar observations were made by Aryantha & Lunggani (2007), who observed that L. plantarum, L. fermentum and Lactobacillus delbrueckii significantly inhibited fungal growth of A. flavus and AFB1 production. Dalié et al. (2010) established that the main LAB recognized for their ability to limit mycotoxinogenic mould growth belong to the genera Lactococcus and Lactobacillus, including L. rhamnosus, in agreement with our results. These results reflect a strong ability to inhibit growth rate and AFB1 production by both Lactobacillus strains with a wide spectrum of antimicrobial activity and high probiotic potential. This suggest that the use of LAB with antifungal properties instead of chemical preservatives would enable the food and feed industry to produce organic food without chemical additives. In addition to the known excellent properties of Lactobacillus strains, they could enhance the nutritional value and prolong the conservation of food. These results are important given that these aflatoxicogenic fungi are natural contaminants of raw materials used for food and feed production, which could be effectively controlled by L. rhamnosus L60 and L. fermentum L23, both strains having probiotic properties. It is concluded that, under favourable conditions, the two lactobacilli strains not only inhibited aflatoxicogenic fungal growth, but also inhibited AFB1 biosynthesis. Future studies with L. rhamnosus L60 and L. fermentum L23 may test the application of these lactobacilli as biocontrollers of fungal contaminants and also to extend the self life of food and feed stuffs, approaching in situ their probiotic properties.