The Use of Sodium Chloride for Determining Viable Mold Counts and Mold Flora in Foods.

The incorporation of 7.5% NaCl into potato dextrose agar (PDA), a medium routinely used for determining viable mold counts in foods, was effective in inhibiting fast-growing "spreader" molds. Viable mold counts using PDA with and without 7.5% NaCl were determined for 361 test samples of 12 food types, including dry, fleshy and frozen fleshy products. Only PDA plus 7.5% NaCl effectively inhibited the spreaders; food type did not influence its effectiveness. Average viable mold counts were usually higher for the 12 food types on PDA containing NaCl. With few exceptions, NaCl did not inhibit nonspreaders; in some cases it enhanced detection of more species, including several mycotoxin producers.

Effect of Other Toxigenic Mold Species on Aflatoxin Production by Aspergillus flavus in Sterile Broth Shake Culture.

The effect of Aspergillus ochraceus , A. versicolor , Penicillium citrinum , P. cyclopium and P. urticae on production of aflatoxin by A. flavus when grown together with A. flavus in rotary shake culture was investigated. The two aspergilli had no apparent effect on aflatoxin production, whereas all three Penicillium species substantially lowered aflatoxin production. The toxins that these penicillia produced when growing in pure culture were not found when the penicillia were grown with A. flavus . However, these toxins had no effect on aflatoxin production added to the growth media, nor did the three molds metabolize aflatoxin. When A. flavus was grown in both filter- and autoclave-sterilized filtrates of these three species, no aflatoxins were produced, although A. flavus grew well. These results suggest that although A. ochraceus and A. versicolor have no apparent effect on aflatoxin production, P. citrinum , P. cyclopium and P. urticae produce heat-stable, nonfilterable metabolite(s) which inhibit(s) aflatoxin production by actively growing A. flavus .

Effect of Temperature, Water Activity and Other Toxigenic Mold Species on Growth of Aspergillus flavus and Aflatoxin Production on Corn, Pinto Beans and Soybeans.

Portions of corn, a commodity in which aflatoxin is frequently found, were held at 16, 26 and 32°C after the moisture contents were adjusted to achieve water activities (aw) ranging from too low to ample for support of mold growth. Suspensions of mold spores from toxigenic cultures of Aspergillus flavus , A. ochraceus , Penicillium citrinum , P. cyclopium and P. urticae were added to the test portions, either as A. flavus alone, as A. flavus with one of the other molds or as a mixture of all 5 species. Additional water was used to obtain the proper moisture levels. A temperature of 16°C was generally too low for aflatoxin production by either the added or native strains of A. flavus , although the mold was able to grow at 16°C at aw values as low as 0.80, 0.77 and 0.85 on corn, soybeans and pinto beans, respectively. Aflatoxin production was essentially the same at 26 and 32° C with limiting aw values in the range of 0.85-0.89. Limiting aw values for mold growth at 26 and 32°C were 0.73, 0.69 and 0.75 for corn, soybeans and pinto beans, respectively. This study provided no evidence that substrate suitability at limiting temperatures and aw levels is a factor in the observed difference in the risk of aflatoxin contamination for these commodities. The study did indicate that the associated mold flora, when the seed is exposed to mold invasion, is a risk determinant.

Molds and Tenuazonic Acid in Fresh Tomatoes Used for Catsup Production.

The mold flora was determined for 146 samples of fresh but visibly moldy tomatoes collected from sorting belts in tomato catsup processing plants in California and in Midwestern and Eastern United States. Mold found in 141 of the samples included at least 22 genera, principally Alternaria , Aspergillus , Cladosporium , Fusarium and Penicillium , and 51 species. The California tomatoes were dominated by Geotrichum candidum and species of Aspergillus and Penicillium ; Midwest and East tomatoes were dominated by Alternaria . This suggested that the predominant molds in tomatoes may differ, depending on geographical source. Tenuazonic acid (TA), a toxic metabolite of Alternaria spp., was found in 73 of the samples at a range of 0.4 to 69.7 (average 4.94) µg/g of moldy tissue; however, Alternaria spp. were not found in 35 of the 73 TA-positive samples. It is possible that other molds may produce TA or that the toxin-producing Alternaria died off before our sampling.

Effects of Processing Equipment on Howard Mold and Rot Fragment Counts of Tomato Catsup.

Two studies were done to determine the effects of processing equipment on Howard mold and rot fragment counts of tomato catsup. In a pilot plant study in 1980, batches of catsup with known cut-out rot levels were produced and processed through various types of comminution equipment. Urschel and Fitzpatrick mills and homogenizers at 500 to 700 and 1500 to 2000 psi increased mold counts more than twofold over the range of data obtained. Contrary to previous reports, Urschel mills increased rot counts significantly. A nationwide survey was conducted in 1983 to determine if similar effects would be found with well-characterized commercial products. Data were obtained on inline and finished products from 164 lots of catsup produced at 16 plants located across the country. Urschel and Fitzpatrick mills tended to increase mold counts over twofold and caused a slight increase in rot counts. High pressure homogenizers (≥2000 psi) tended to decrease mold counts; low pressure homogenizers (<2000 psi) increased them. Homogenization at any pressure reduced rot counts dramatically. Although mold counts were highest for catsup produced in the eastern United States and lowest for catsup produced in the West, milling and low pressure homogenization were also most prevalent in the East and least prevalent in the West. When the effects of these types of comminution were removed, the difference between regions diminished. Compared with the norm, rainfall levels for the growing regions involved in this survey were fairly typical.

Ochratoxins A and B, Xanthomegnin, Viomellein and Vioxanthin Production by Isolates of Aspergillus ochraceus from Green Coffee Beans.

Isolates from Aspergillus ochraceus obtained from green coffee beans were cultured on rice and water. After 20 d of growth the cultures were extracted with chloroform and the extracts were analyzed by high performance liquid chromatography for ochratoxin A (OA), ochratoxin B (OB), xanthomegnin (X), viomellein (V) and vioxanthin (VX). Forty-three percent of the isolates produced OA at an average level of 397 µg of toxin/g rice, 17% produced OB at an average level of 312 µg/g, and 84% produced X, V, and VX at an average level of 281, 417 and 386 µg/g, respectively. The highest levels of toxin production were OA, 2088 µg/g; OB, 3375 µg/g; X, 1562 µg/g; V, 2514 µg/g; and VX, 2054 µg/g. VX has not previously been reported as an A. ochraceus metabolite.

Incidence of Toxigenic and Other Molds in Green Coffee Beans.

The mold flora of 944 green coffee bean samples from 31 coffee-producing countries was determined before and after surface disinfection with 5% NaOCl. Molds were detected on 99.1 % of 47,200 beans not surface-disinfected and in 47.9% of 47,200 disinfected beans. Although the percentage of differences in occurrence of mold before disinfection was minimal (93.4 to 100%) on a country-by-country basis, after disinfection the beans from Asiatic and African countries showed more internal invasion (80.5%) than those from Central and South America (49.4%). Aspergillus spp., which dominated the mold flora of 944 samples before and after disinfection, included the toxigenic A. ochraceus , A. flavus and A. versicolor as well as A. niger , A. tamarii , A. wentii and species of the A. glaucus group. The genus Penicillium , including the toxigenic P. cyclopium , P. citrinum and P. expansum , was detected regularly, although its occurrence was substantially lower than that of the aspergilli, especially after surface disinfection. The rare detection of Alternaria and Fusarium indicated that toxigenic species of these genera do not readily invade green coffee beans. A. flavus and A. tamarii were prevalent in Central and South American beans, whereas other aspergilli were prevalent in Asiatic and African beans. The penicillia were prevalent in Central and South American beans.

Toxic Species of Penicillium Common in Food.

Brief descriptions are presented of six toxigenic Penicillium species: P. cyclopium , P. viridicatum , P. islandicum , P. expansum , P. citrinum and P. urticae . The profiles are based on previous studies in which mold flora were determined in various stored and dried foods. The toxic metabolites of these Penicillium species are also reviewed.

Preparation of 14C-Labeled Penicillic Acid with High Specific Activity and Yield.

14C-Labeled penicillic acid was produced by stationary culture incubation of Penicillium cyclopium (NRRL 1888) on a modified Raulin-Thom broth medium containing 14C-labeled acetate. Approximately 1.2 g of radioactive compound, with a specific activity of 23.0 µCi/mmole, was produced in 9 days in 1500 ml of the broth. Incorporation of the isotope into penicillic acid was 11. 9%. Production of the radiolabeled compound with high specific activity was achieved by correlating the monitoring of expired 14C-CO2 with production of penicillic acid during the fermentation. The effects of various growth substrates, pH, and incubation times on production of non-labeled penicillic acid also were investigated. Results show that sterile rice is an excellent substrate, that among liquid media examined, higher yields were obtained in stationary rather than in shake cultures, and that higher yields of penicillic acid were obtained at pH 3.5 or lower. Simultaneous monitoring of penicillic acid production and 14C-label incorporation is essential to detect and isolate a high yield of labeled compound with high specific activity.

Incidence of Toxic and Other Mold Species and Genera in Soybeans.

The mold flora of 385 unprocessed soybean samples was determined before and after surface disinfection of the beans with 5% NaOCl. Molds were detected in 99.4% of the 19,250 non-disinfected soybeans examined, and in 52.8% of the 19,250 surface-disinfected soybeans examined indicating that contamination was primarily at the surface, although internal invasion also was substantial. The mold flora, both before and after surface disinfection, was dominated by species of the Aspergillus glaucus group, Asperigillus flavus , Aspergillus candidus , Penicillium cyclopium , plus species of Alternaria and Cladosporium . The toxicogenic species A. flavus , Aspergillus nidulans , Aspergillus ochaeceus , Aspergillus versicolor , Penicillium citrinum , P. cyclopium , and Pencillium viridicatum were encountered regularly in non-surface-disinfected soybeans. Except for A. flavus and P. cyclopium , the occurrence of toxicogenic species Aspergillus melleus , soybeans was sporadic. The toxicogenic species Aspergillus melleus , Pencillium expansum , and Penicillium urticae were encountered occasionally, but only in non-surface-disinfected soybeans. Penicillium chrysogenum , the penicillin-producing organism, was encountered regularly in non-surface-disinfected soybeans, but less frequently in disinfected soybeans.