Aspergillus ochraceus growth and ocratoxin A production inhibited by natural phenolic compounds and weak organic acids

none4Prevention of fungal growth is becoming increasingly interesting in connection with the prevention of mycotoxin contamination in several foods. To this purpose, natural compounds represent possible alternatives to most food additives currently in use. Ochratoxins are regarded as powerful nephrotoxins, carcinogens, teratogens, and immunotoxins in rats and likely also in humans (group 2B). Among them ochratoxin A (OTA) is the most toxic and is produced by a variety of moulds included in the genera Aspergillus (mainly A. ochraceus) that has been detected frequently in cereal products. Naturally occurring preservatives are plant phenolic compounds and weak organic acids that make the cell membrane of fungi permeable. Phenolic compounds, such as thymol and carvacrol, are responsible for the antimicrobial properties of many essential oils and appear to act as membrane permeabilizers. The antimicrobial activity of lipophilic weak-acids is well documented and they appear to share a common mode of action strongly pH-dependent involving their diffusion through the plasma membrane into the cytoplasm. Rarely, the microorganisms affected by those AM are killed, but their growth is prevented. Carvacrol and thymol, and three organic acids (sorbic, citric, and propionic acid) were used as antimicrobials (AM) to study their influence on the growth and on OTA production by the Aspergillus ochraceus (Ao) 5137. The effects of AM on the growth was evaluated either in YES broth and in agar plates. Thymol and carvacrol stably inhibited the growth in YES broth at concentration ≥0.66 mM. In YES agar plates, 6 days after the inoculation, the % of growth inhibition induced by sorbic, citric and propionic acids was respectively equal to 80.20%, 69.90% and 29.55%. In the control the maximum growth was reached after 8 days whereas in the samples treated with sorbic, citric and propionic acids the % of inhibition was respectively equal to 67.9%, 61.0% and 15%. To evaluate the effects of AM on OTA production after 12, 15 and 20 days of incubation, portions of YES broth were cleaned-up with Ochraprep columns. The OTA concentration was determined by HPLC. At the end of each trial the mycelial dry weight was determined. The production of OTA by Ao on YES broth was significantly (P < 0.05) inhibited by all the AM treatments as compared to the untreated controls after 12, 15, and 20 d of incubation. In particular, after 20 d of fungal growth, sorbic acid 31.2 mM reduced the level of OTA production by 0.3% than control (0.25 ± 0.003 and 100.00 ± 50.91 ppm, respectively; P < 0.01) whereas thymol and carvacrol 0.5 mM reduced the level by 71.3% and 30.7% respectively. Finally, citric (18 mM), sorbic (31.2 mM) and propionic acids (26.8 mM) constrained the OTA level by 85.0%, 99.8% and 99.1% respectively, after 20 d of growth. These data showed that other than sorbic acid, that clearly reduce proximal to zero the OTA production, the other AM tested could be considered efficient only in delaying toxin production. A relationship between mycotoxin production and sporulation in the presence of the AM tested will be discussed.noneMinardi P.; Pizzamiglio V.; Mucini S.; Piva A.Minardi P.; Pizzamiglio V.; Mucini S.; Piva A