Efficiency of Hydroxycinnamic Phenolic Acids to Inhibit the Production of Ochratoxin A by Aspergillus westerdijkiae and Penicillium verrucosum

Ochratoxin A (OTA) is one of the worldwide most important mycotoxins in terms of health and agroeconomic consequences. With the aim to promote the use of phytochemicals as alternatives to synthetic fungicides, the effect of hydroxycinnamic acids on the fungal growth and OTA yield by two major OTA-producing species was investigated. After a first step dedicated to the definition of most suitable culture conditions, the impact of 0.5 mM ferulic (FER), p-coumaric (COUM), caffeic and chlorogenic acids was evaluated on Aspergillus westerdijkiae and Penicillium verrucosum. Whereas no fungal growth reduction was observed regardless of the phenolic acid and fungal isolate, our results demonstrated the capacity of FER and COUM to inhibit OTA production. The most efficient compound was FER that led to a 70% reduction of OTA yielded by P. verrucosum and, although not statistically significant, a 35% inhibition of OTA produced by A. westerdijkiae. To further investigate the bioactivity of FER and COUM, their metabolic fate was characterized in fungal broths. The capacity of P. verrucosum to metabolize FER and COUM through a C2-clivage type degradation was demonstrated. Overall, our data support the potential use of FER to prevent OTA contamination and reduce the use of synthetic pesticides.

Effect of a Debaryomyces hansenii and Lactobacillus buchneri Starter Culture on Aspergillus westerdijkiae Ochratoxin A Production and Growth during the Manufacture of Short Seasoned Dry-Cured Ham

Recently, specific dry-cured hams have started to be produced in San Daniele and Parma areas. The ingredients are similar to protected denomination of origin (PDO) produced in San Daniele or Parma areas, and include pork leg, coming from pigs bred in the Italian peninsula, salt and spices. However, these specific new products cannot be marked as a PDO, either San Daniele or Parma dry cured ham, because they are seasoned for 6 months, and the mark PDO is given only to products seasoned over 13 months. Consequently, these products are called short-seasoned dry-cured ham (SSDCH) and are not branded PDO. During their seasoning period, particularly from the first drying until the end of the seasoning period, many molds, including Eurotium spp. and Penicillium spp., can grow on the surface and work together with other molds and tissue enzymes to produce a unique aroma. Both of these strains typically predominate over other molds. However, molds producing ochratoxins, such as Aspergillus ochraceus and Penicillium nordicum, can simultaneously grow and produce ochratoxin A (OTA). Consequently, these dry-cured hams may represent a potential health risk for consumers. Recently, Aspergillus westerdijkiae has been isolated from SSDCHs, which could represent a potential problem for consumers. Therefore, the aim of this study was to inhibit A. westerdijkiae using Debaryomyces hansenii or Lactobacillus buchneri or a mix of both microorganisms. Six D. hansenii and six L. buchneri strains were tested in vitro for their ability to inhibit A. westerdijkiae. The strains D. hansenii (DIAL)1 and L. buchneri (Lb)4 demonstrated the highest inhibitory activity and were selected for in situ tests. The strains were inoculated or co-inoculated on fresh pork legs for SSDCH production with OTA-producing A. westerdijkiae prior to the first drying and seasoning. At the end of seasoning (six months), OTA was not detected in the SSDCH treated with both microorganisms and their combination. Because both strains did not adversely affect the SSDCH odor or flavor, the combination of these strains are proposed for use as starters to inhibit OTA-producing A. westerdijkiae.

Resilience of Aspergillus westerdijkiae Strains to Interacting Climate-Related Abiotic Factors: Effects on Growth and Ochratoxin A Production on Coffee-Based Medium and in Stored Coffee

We examined the resilience of strains of Aspergillus westerdijkiae in terms of growth and ochratoxin A (OTA) production in relation to: (a) two-way interacting climate-related abiotic factors of water activity (aw, 0.99–0.90) × temperature (25–37 °C) on green coffee and roasted coffee-based media; (b) three-way climate-related abiotic factors (temperature, 30 vs. 35 °C; water stress, 0.98–0.90 aw; CO2, 400 vs. 1000 ppm) on growth and OTA production on a 6% green coffee extract-based matrix; and (c) the effect of three-way climate-related abiotic factors on OTA production in stored green coffee beans. Four strains of A. westerdijkiae grew equally well on green or roasted coffee-based media with optimum 0.98 aw and 25–30 °C. Growth was significantly slower on roasted than green coffee-based media at 35 °C, regardless of aw level. Interestingly, on green coffee-based media OTA production was optimum at 0.98–0.95 aw and 30 °C. However, on roasted coffee-based media very little OTA was produced. Three-way climate-related abiotic factors were examined on two of these strains. These interacting factors significantly reduced growth of the A. westerdijkiae strains, especially at 35 °C × 1000 ppm CO2 and all aw levels when compared to 30 °C. At 35 °C × 1000 ppm CO2 there was some stimulation of OTA production by the two A. westerdijkiae strains, especially under water stress. In stored green coffee beans optimum OTA was produced at 0.95–0.97 aw/30 °C. In elevated CO2 and 35 °C, OTA production was stimulated at 0.95–0.90 aw.

Antifungal Activity of Selected Natural Preservatives against Aspergillus westerdijkiae and Penicillium verrucosum and the Interactions of These Preservatives with Food Components

ABSTRACT The present study examined the influence of primary food components on the antifungal activity of the essential oil of Origanum vulgare, carvacrol, thymol, eugenol, and trans-cinnamaldehyde against Penicillium verrucosum and Aspergillus westerdijkiae. The MIC was determined in food model media enriched with proteins (1, 5, or 10%), carbohydrates (1, 4, or 6%), or oil (1, 5, or 10%). Proteins increased the antifungal activity of O. vulgare essential oil, carvacrol, thymol, and eugenol, whereas the effect of trans-cinnamaldehyde decreased with increasing protein content. The presence of carbohydrates diminished the inhibitory effect of the natural preservatives on A. westerdijkiae; for P. verrucosum, their inhibitory effect increased with carbohydrates. Only the antifungal activity of trans-cinnamaldehyde did not depend on the carbohydrate content. The presence of oil had the strongest influence. At a concentration of 1% oil, the antifungal activity decreased significantly, and at 10% oil, almost no inhibition was observed. To investigate the effect of the antifungal agents on the morphology of the target molds, they were grown on malt extract agar containing carvacrol and trans-cinnamaldehyde and were examined by scanning electron microscopy. The hyphae, conidiophores, vesicles, and phialides were severely altered and deformed, and spore formation was clearly suppressed.