Ochratoxigenic potential of Aspergillus westerdijkiae NRRL 3174 under laboratory conditions

The family of ochratoxins consists of three members known as ochratoxin A, ochratoxin B and ochratoxin C. They are the second major group of mycotoxins to be characterized after the discovery of aflatoxins. Structurally, the three toxins differ only very slightly from each other; however, these differences have marked effects on their respective toxic potentials, with ochratoxin A being the most toxic of that family based on the median lethal dose and minimal growth inhibition in birds (Peckham et al., 1971; Chang et al., 1979). OTA is produced by fungi from two genera, Aspergillus and Penicillium. There is the general opinion that species of Aspergillus genus are important in OTA production in warmer regions, while in colder regions, especially in temperate climates, only the activity of Penicillium verrucosum is significant (Elmholt and Hestbjerg, 1999; Lindblad et al., 2004; Park et al., 2005). Moisture content and temperature are the most important variables in determining growth and rate of mycotoxin production by fungi in stored grain ecosystem (Cairns-Fuller et al., 2005; Pardo et al., 2004). Various other fungal species present on grain differ in their growth responses to temperature and water activity of the substrate (Haasum and Nielsen, 1998; Pardo et al., 2004; Ramakrishna et al., 1996). The study was carried out to determine the ochratoxigenic potential of Aspergillus westerdijkiae NRRL 3174 as influenced by sucrose concentrations in liquid medium and different temperatures, moisture and incubation period in solid substrates under laboratory conditions.

The lyophilised preparation of Aspergillus westerdijkiae NRRL 3147 was obtained from U.S. Department of Agriculture, Peoria, Illinois (U.S.A.). This lyophilized preparation was revived on potato dextrose agar medium and used for experimentation. Two experiments were conducted in this study. In experiment one, the medium consisted of 2% yeast extract and various concentrations of sucrose ranging from 0 to 16 percent was used and the inoculum was Aspergillus westerdijkiae NRRL 3174. This experiment was conducted to establish the optimum concentration of sucrose for maximum production of ochratoxin A.

The yeast extract- sucrose medium was incubated at 25±0.5oC for two weeks in Roux flasks in a REMI make BOD incubator. The experiment was replicated thrice and the yields were reported as averages. The final pH of the medium was determined. Cultures were filtered and the dry weight of mycelium was determined after drying the mycelial mats overnight at 70oC in a hot air oven. Ochratoxin A was extracted from culture filtrates using chloroform in separatory funnels. The chloroform extracts were then evaporated to dryness on a steam bath. One ml of chloroform was then added to the dry samples. OTA assays were performed by thin layer chromatography (TLC) by spotting few microliters of the samples on silica gel G prepared TLC plates. TLC plates were developed in toluene-ethyl acetate-formic acid (5:4:1; v/v) as reported by Scott and Hand (1967). The plates were examined under ultraviolet light and the samples were diluted or concentrated until the fluorescent intensities of the sample and the standard were the same. The OTA was determined quantitatively by visual comparison with OTA standard.

In second experiment, the effect of moisture, temperature and length of incubation period on OTA production using solid substrate namely maize and rice was studied. Fifty grams of cracked maize or broken rice were taken separately in 250 ml conical flask. The moisture content of each substrate was adjusted to have a moisture level of 15, 20, 25, 30, and 35 percent. Thus, a total of 80 flasks (40 for maize and 40 for rice substrate) were plugged with non-absorbent cotton and sealed with aluminium foil. The flasks were autoclaved for 20 minutes at 121oC and inoculated with one week old spores of Aspergillus westerdijkiae NRRL 3174. The inoculated flasks were incubated in a BOD incubator for a period of 7, and 14 days. The same experiment was repeated at 20, 25, 30 and 35oC temperature. After removal from the incubator, the flasks were dried at 70oC and the ochratoxin A assays were performed as per AOAC (1995).

The effect of sucrose concentration on ochratoxin A production by Aspergillus westerdijkiae is given in Table 1.

The fungal growth continued to increase with increasing concentrations of sucrose from 0 to 16 percent. Thus, the mycelia dry weight was the lowest (0.21 g/100 ml) at 0% concentration and highest (3.65 g/100 ml) at 16% sucrose concentration. By and large, the final pH of the medium showed a decreasing trend with the increase in sucrose concentration and it was below 7 when the sucrose concentration was 8% or higher. The highest production of ochratoxin A (21 mg/100 ml) was recorded at 8% sucrose concentration and 2% yeast extract medium. Davis et al. (1969) reported that peak ochratoxin A production (29 mg/100ml) occurred in 4% sucrose and 2% yeast extract concentration.

In present study, the ochratoxin A production increased with the increase in sucrose concentration up to 8%. However, above 8% sucrose concentration, a decrease in ochratoxin A production was recorded. Therefore, ochratoxin A production is neither related to fungal growth nor the sucrose concentration in the medium. Davis et al. (1969) also reported that the fungal growth continued to increase directly with higher concentrations of sucrose from 4 to 32% without a corresponding increase in ochratoxin A production.

Table 1. Effect of sucrose concentration on OTA production by Aspergillus westerdijkiae in 2% yeast extract solution

Ochratoxigenic potential of Aspergillus westerdijkiae NRRL 3174 under laboratory conditions - Image 1

The effect of moisture, temperature and length of incubation period on Ochratoxin A production on cracked maize by Aspergillus westerdijkae NRRL 3174 is recorded in Table 2. The ochratoxin A yield increased with the increase in moisture content from 15 to 35%. Up to 25% moisture level, very low yield of ochratoxin A was recorded, however, considerably higher yield of ochratoxin A was recorded at 30 and 35% moisture level. The highest yield of ochratoxin A was recorded at 35% moisture level. These findings suggest that a minimum of 35% moisture level is required for maximum production of ochratoxin A by Aspergillus westerdijkiae NRRL 3174.

In 7 days of incubation period, the highest production of ochratoxin A (0.88 g/kg) was recorded at 35% moisture and 25oC temperature followed by 0.81 g/kg maize at 35% moisture and 30oC temperature. In 14 days of incubation period, the highest production of ochratoxin (0.99 g/kg maize) was recorded at 35% moisture and 30oC temperature followed by 0.92 g/kg maize at 35% moisture and 25oC temperature. Considerably lower yields of ochratoxin A were recorded at 20 and 35oC as compared to 25 and 30oC temperatures. Thus, the optimum temperature for maximum production of ochratoxin A by Aspergillus westerdijkiae NRRL 3174 lies between 25 and 30oC. Trenk et al. (1971) also reported that the optimal temperature for ochratoxin A production by Aspergillus ochraceus NRRL 3174 was found to be around 28oC.

The effect of moisture, temperature and length of incubation period on Ochratoxin A production on broken n rice by Aspergillus westerdijkae NRRL 3174 is recorded in Table 3. The ochratoxin A yields increased with the increase in moisture content from 15 to 35% and the highest yield of ochratoxin A was recorded at 35% moisture level. Almost similar trend in the production of ochratoxin A has been observed as in the case of maize.

Table 2. Effect of moisture, temperature and incubation period on ochratoxin production in maize by Aspergillus westerdijkiae.

Ochratoxigenic potential of Aspergillus westerdijkiae NRRL 3174 under laboratory conditions - Image 2

In seven days of incubation period, the highest production of ochratoxin A (0.98 g/kg rice) was recorded at 35% moisture and 30oC temperature followed by 0.95 g ochratoxin /kg rice at 35% moisture and 25oC temperature. In 14 days of incubation period, the highest production of ochratoxin A (0.98 g/kg rice) was recorded at 35% moisture and 300C temperature followed by 0.98 g ochratoxin /kg rice at 35% moisture and 30oC temperature. As in the case of maize, considerably lower yields of ochratoxin A were recorded at 20 and 35oC as compared to 25 and 30oC temperature. Harwig and Chen (1974) did not observe any symptoms of growth of Penicillium viridicatum and OTA synthesis by the fungus after two weeks of incubation at 12oC temperature and 18% moisture content, but the fungus produced OTA at 12oC and 22% moisture level. Cairns-Fuller et al. (2005) also found that Scandinavian strains of Penicillium verrucosum grew and produced OTA on wheat grain at moisture level equal to or greater than 19%. The best conditions for fungal growth and OTA formation in their studies were 25% moisture content and 25oC temperature.

It is thus concluded that fungal growth is not related to ochratoxin production and the maximum yield of OTA can be harvested at 8% sucrose and 2% yeast extract medium. Further, the maximum yield of OTA on solid substrate can be harvested at 35% moisture level and between 25 and 30oC temperature.

This article was originally published in Indian Journal of Poultry Science (2013) 48(2): 247-24.

Ochratoxigenic potential of Aspergillus westerdijkiae NRRL 3174 under laboratory conditions

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