Comparison of dichloran 18% glycerol (DG18) agar with general purpose mycological media for enumerating food spoilage yeasts

Dichloran 18% glycerol (DG18) agar was originally developed to enumerate xerophilic foodborne moulds. However, some laboratories are using DG18 agar as a general medium to enumerate foodborne moulds and yeasts. A collaborative study, with the participation of seven laboratories, was undertaken to compare DG18 agar with dichloran rose bengal chloramphenicol (DRBC) agar, tryptone glucose yeast extract chloramphenicol (TGYC) agar, and plate count agar supplemented with chloramphenicol (PCAC) for enumerating 14 species of common food spoilage yeasts. Comparison of the mean values of populations of all yeasts recovered on each medium revealed no significant differences among DRBC agar, PCAC, and TGYC agar, while each of these media supported the development of significantly (P < or = 0.05) higher numbers of colonies than DG18 agar. However, differences were only 0.08 to 0.10 log10 cfu/ml, making the practical significance questionable. The overall coefficient of variation (CV) for within laboratory repeatability was 1.71%, while the CV for reproducibility of counts obtained among laboratories was 6.96%. Compared to DRBC agar, TGYC agar, and PCAC, yeast colonies were smaller on DG18 agar. Growth of Brettanomyces anomalus, Cryptococcus albidus, and Rhodotorula mucilaginosa was particularly retarded or inhibited on DG18 agar. Based on the performance of media in supporting colony development and ease of counting colonies, the use of DG18 agar as a general enumeration medium for foodborne yeasts cannot be recommended.     

AFLATOXIGENIC ASPERGILLI IN FOODS AND FEEDS IN UGANDA

Studies conducted during the sixties and the seventies on food crops in Uganda showed that the populace was exposed to consumption of aflatoxin-contaminated foods. These studies also linked the highest incidence of liver cancer in the world to the presence of high levels of aflatoxins in the food and beverages. After a lapse of a decade, it was of interest to investigate the occurrence of aflatoxins and aflatoxigenic fungi in staple Ugandan food crops and poultry feeds derived from these foodstuffs. A simple, rapid and reproducible procedure was used. The procedure consisted of growing or culturing feed grains on a selective medium, Aspergillus flavus/parasiticus agar (AFPA) followed by screening for aflatoxin producing fungi on a coconut agar medium (CAM) under UV light with a subsequent confirmatory screening method for aflatoxin production by the fungi in pure culture. Fifty-four samples consisting of corn and peanuts, soybean and poultry feed were analyzed for content of aflatoxigenic. A. flavus/parasiticus and 25 of the samples were also screened for aflatoxins B₁ and G₁, zearalenone, sterigmatocystin, ochratoxin A, citrinin, vomitoxin, and diacetoxyscirpenol (DAS). Aflatoxigenic A. flavus/parasiticus was detected from the majority of corn (77%), peanuts (36% human food and 83.3% animal feed) and poultry feed (66.6%). but not from soybean samples. Two samples out of 25 contained detectable levels of aJatosin B, (20 ppb). For the jirst time other mycotoxins, zearalenone (3 samples) and vomitoxin (2 samples) were detected in corn from Uganda.     

INCIDENCE OF AFLATOXIGENIC ISOLATES OF ASPERGILLUS FLAVUS/PARASITICUS OBTAINED FROM GEORGIA CORN PROCESSING PLANTS

Two corn processing facilities within Georgia were evaluated in order to determine the incidence of Aspergillus flavus or A. parasiticus within the plant and in corn harvested and processed in 1984 and 1985. Conidia of A. flavus/parasiticus were found in all corn samples evaluated as well as in settled dust samples taken within these processing facilities. Isolates were obtained by using the differential/selective medium Aspergillus flavus/parasiticus agar. Upon subsequent culture only 55% of the selected isolates were confirmed as belonging to A. flavus/parasiticus group. Some of these isolates were randomly chosen and their ability to produce aflatoxins B₁, B₂, G₁, or G₂ evaluated. Thirty-two percent of the A. flavus/parasiticus isolates cultured for aflatoxin production were found to be aflatoxigenic.     

Aqueous extracts of Tulbaghia violacea inhibit germination of Aspergillus flavus and Aspergillus parasiticus conidia

Aspergillus flavus and Aspergillus parasiticus are important plant pathogens and causal agents of pre- and postharvest rots of corn, peanuts, and tree nuts. These fungal pathogens cause significant crop losses and produce aflatoxins, which contaminate many food products and contribute to liver cancer worldwide. Aqueous preparations of Tulbaghia violacea (wild garlic) were antifungal and at 10 mg/ml resulted in sustained growth inhibition of greater than 50% for both A. flavus and A. parasiticus. Light microscopy revealed that the plant extract inhibited conidial germination in a dose-dependent manner. When exposed to T. violacea extract concentrations of 10 mg/ml and above, A. parasiticus conidia began germinating earlier and germination was completed before that of A. flavus, indicating that A. parasiticus conidia were more resistant to the antifungal effects of T. violacea than were A. flavus conidia. At a subinhibitory extract dose of 15 mg/ml, hyphae of both fungal species exhibited increased granulation and vesicle formation, possibly due to increased reactivity between hyphal cellular components and T. violacea extract. These hyphal changes were not seen when hyphae were formed in the absence of the extract. Transmission electron microscopy revealed thickening of conidial cell walls in both fungal species when grown in the presence of the plant extract. Cell walls of A. flavus also became considerably thicker than those of A. parasiticus, indicating differential response to the extract. Aqueous preparations of T. violacea can be used as antifungal treatments for the control of A. flavus and A. parasiticus. Because the extract exhibited a more pronounced effect on A. flavus than on A. parasiticus, higher doses may be needed for control of A. parasiticus infections.     

THE AFLATOXIN CONTAMINATION OF FIG FRUITS IN AYDIN CITY (TURKEY)

The mold flora of 50 dried fig samples consumed in Turkey was examined and the aflatoxigenic ones were determined. Among 127 fungi isolated, 74 were Aspergillus, 24 were Trichoderma, 16 were Fusarium and 13 were Acremonium. Of the isolates, 17 were aflatoxigenic and four of them were capable to produce aflatoxin, three of which were characterized as A. flavus and one as A. parasiticus. Aflatoxin production of four strains was confirmed by high pressure liquid chromotography. The effect of UV irradiation on mold count and aflatoxin quantity was also tested. It was found that UV irradiation led to a decrease in the mold count and aflatoxin quantity.

PRACTICAL APPLICATIONS

Studies have shown that the concentration of aflatoxins may exceed the determined limits in dried figs. Its presence can be a potential threat to the health of consumers. Dried figs are one of the major agricultural export products of Turkey ( Senyuva et al. 2005 ). The effects of UV irradiation on mold flora of dried figs and aflatoxins have been examined. The Aspergillus flavus and parasiticus agar (AFPA) medium is used for detection of aflatoxigenic species, and coconut agar medium (CAM) is used to detect the aflatoxin-producing ability of aflatoxigenic strains. It was found that the reproduction of the molds in dried figs, consequently the aflatoxigenic mold strains, can be depressed by UV irradiation. It was found that increasing time of UV irradiation led to a decrease in the mold count in dried figs. In addition, a UV irradiation applied for 90 min, was found to decrease the aflatoxin quantity in dried figs in an amount of 25%. Because of inexpensiveness and easiness of the application it was concluded that the UV irradiation can be used as a practical application.     

Comparison of dry sheet media and conventional agar media methods for enumerating yeasts and molds in food

A study was done to compare Nissui Compact Dry Yeast and Mold plates (CDYM), 3M Petrifilm Yeast and Mold count plates (PYM), dichloran-rose bengal chloramphenicol (DRBC) agar, and dichloran 18% glycerol (DG18) agar for enumerating yeasts and molds naturally occurring in 97 foods (grains, legumes, raw fruits and vegetables, nuts, dairy products, meats, and miscellaneous processed foods and dry mixes). Correlation coefficients for plates incubated for 5 days were DG18 versus DRBC (0.93), PYM versus DRBC (0.81), CDYM versus DG18 (0.81), PYM versus DG18 (0.80), CDYM versus DRBC (0.79), and CDYM versus PYM (0.75). The number of yeasts and molds recovered from a group of foods (n = 32) analyzed on a weight basis (CFU per gram) was not significantly different (alpha = 0.05) when samples were plated on DRBC, DG18, PYM, or CDYM. However, the order of recovery from foods (n = 65) in a group analyzed on a unit or piece basis, or a composite of both groups (n = 97), was DRBC > DG18 = CDYM > PYM. Compared with PYM, CDYM recovered equivalent, significantly higher (alpha = 0.05) or significantly lower (alpha = 0.05) numbers of yeasts and molds in 51.5, 27.8, and 20.6%, respectively, of the 97 foods tested; respective values were 68.8, 15.6, and 15.6% in the small group (n = 32) and 43.1, 33.8, and 23.1% in the large group (n = 65) of foods. The two groups contained different types of foods, the latter consisting largely (73.8%) of raw fruits (n = 16) and vegetables (n = 32). Differences in efficacy of the four methods in recovering yeasts and molds from foods in the two groups are attributed in part to differences in genera and predominant mycoflora. While DG18 agar, CDYM, and PYM appear to be acceptable for enumerating yeasts and molds in the foods analyzed in this study, overall, DRBC agar recovered higher numbers from the 97 test foods, thereby supporting its recommended use as a general purpose medium for mycological analysis.     

Recovery of Listeria monocytogenes on selective agar media in a collaborative study using reference samples

Sixteen laboratories compared counts of Listeria monocytogenes in reference samples using Blood agar, Palcam(y) agar and Oxford agar. Significant differences were found between laboratories. The mean counts on Blood agar were significantly higher than on Palcam(y) or Oxford agar. The mean counts on Palcamy agar were somewhat higher than on Oxford agar (only after 48 h incubation), but no significant difference was found. Addition of egg yolk to Palcam agar seems to be beneficial for the recovery of sublethally injured cells. Recovery of L. monocytogenes was higher after 48 h incubation for all media tested.     

Nannocystis exedens: a potential biocompetitive agent against Aspergillus flavus and Aspergillus parasiticus.

This study examined the potential for controlling toxigenic Aspergillus flavus and Aspergillus parasiticus by biological means using a myxobacterium commonly found in soil. The ability of Nannocystis exedens to antagonize A. flavus ATCC 16875, A. flavus ATCC 26946, and A. parasiticus NRRL 3145 was discovered. Cultures of aflatoxigenic fungi were grown on 0.3% Trypticase peptone yeast extract agar for 14 days at 28 degrees C. When N. exedens was grown in close proximity with an aflatoxigenic mold, zones of inhibition (10 to 20 mm) developed between the bacterium and mold colony. A flattening of the mold colony on the sides nearest N. exedens and general stunting of growth of the mold colony were also observed. When N. exedens was added to the center of the cross-streak of a mold colony, lysis of the colony by the bacterium was observed after 24 h. Microscopic observations revealed that N. exedens grew on spores, germinating spores, hyphae, and sclerotia of the molds. These results indicate that N. exedens may be a potential biocontrol agent against A. flavus and A. parasiticus.     

Fungal population and distribution of aflatoxigenic fungi in commercial almond powder products

The fungal population and distribution of aflatoxin-producing fungi in 30 samples of imported almond powder products purchased from retail markets were examined in this study. Total counts of fungi ranged from under 1.0 x 10 colony-forming units (CFU)/g to 8.5 x 10(3) CFU/g as determined with the dilution plating technique. The predominant fungi in the mould-contaminated almond samples were Aspergillus niger, A. flavus and the related species, Penicillium, Cladosporium and Rhizopus. Aflatoxin-producing ability in the isolates of A. flavus and related fungi were tested by thin layer chromatography using 2% yeast extract and 15% sucrose broth culture. Four different aflatoxigenic fungi were detected in the isolates; aflatoxins B1 and B2 were produced by some strains of A. flavus and A. parvisclerotigenus, and aflatoxins B1, B2, G1, G2 were produced by all tested strains of A. parasiticus and A. nomius. Identification of the strains was based on morphological and metabolic characters.     

Comparison of culture media, simplate, and petrifilm for enumeration of yeasts and molds in food

The efficacy of three culture media, dichloran rose bengal chloramphenicol (DRBC), dichloran 18% glycerol agar (DG18), and potato dextrose agar (PDA) supplemented with two antibiotics, were compared with the Simplate and Petrifilm techniques for mold and yeast enumeration. The following foods were analyzed: corn meal, wheat flour, cassava flour, bread crumbs, whole meal, sliced bread, ground peanuts, mozzarella cheese, grated parmesan cheese, cheese rolls, orange juice, pineapple pulp, pineapple cake, and mushroom in conserve. Correlation coefficients of DRBC versus PDA and DG18 for recovering total mold and yeast counts from the composite of 14 foods indicated that the three media were generally equivalent. Correlation coefficients for Petrifilm versus culture media were acceptable, although not as good as between culture media. Correlation coefficients of Simplate versus DRBC, DG18, PDA, and Petrifilm for recovering total yeasts and molds from a composite of 11 foods demonstrated that there was no equivalence between the counts obtained by Simplate and other culture media and Petrifilm, with significant differences observed for the most foods analyzed.     

Fungi, aflatoxins, and cyclopiazonic acid associated with peanut retailing in Botswana

Peanuts are important food commodities, but they are susceptible to fungal infestation and mycotoxin contamination. Raw peanuts were purchased from retail outlets in Botswana and examined for fungi and mycotoxin (aflatoxins and cyclopiazonic acid) contamination. Zygomycetes were the most common fungi isolated; they accounted for 41% of all the isolates and were found on 98% of the peanut samples. Among the Zygomycetes, Absidia corymbifera and Rhizopus stolonifer were the most common. Aspergillus spp. accounted for 35% of all the isolates, with Aspergillus niger being the most prevalent (20.4%). Aspergillus flavus/parasiticus were also present and accounted for 8.5% of all the isolates, with A. flavus accounting for the majority of the A. flavus/parasiticus identified. Of the 32 isolates of A. flavus screened for mycotoxin production, 11 did not produce detectable aflatoxins, 8 produced only aflatoxins B1 and B2, and 13 produced all four aflatoxins (B1, B2, G1, and G2) in varying amounts. Only 6 of the A. flavus isolates produced cyclopiazonic acid at concentrations ranging from 1 to 55 microg/kg. The one A. parasiticus isolate screened also produced all the four aflatoxins (1,200 microg/kg) but did not produce cyclopiazonic acid. When the raw peanut samples (n = 120) were analyzed for total aflatoxins, 78% contained aflatoxins at concentrations ranging from 12 to 329 microg/kg. Many of the samples (49%) contained total aflatoxins at concentrations above the 20 microg/kg limit set by the World Health Organization. Only 21% (n = 83) of the samples contained cyclopiazonic acid with concentrations ranging from 1 to 10 microg/kg. The results show that mycotoxins and toxigenic fungi are common contaminants of peanuts sold at retail in Botswana.     

Aflatoxin reduction in corn through field application of competitive fungi.

Soil in corn plots was inoculated with nonaflatoxigenic strains of Aspergillus flavus and A. parasiticus during crop years 1994 to 1997 to determine the effect of application of the nontoxigenic strains on preharvest aflatoxin contamination of corn. Corn plots in a separate part of the field were not inoculated and served as controls. Inoculation resulted in significant increases in the total A. flavus/parasiticus soil population in treated plots, and that population was dominated by the applied strain of A. parasiticus (NRRL 21369). In the years when weather conditions favored aflatoxin contamination (1996 and 1997), corn was predominately colonized by A. flavus as opposed to A. parasiticus. In 1996, colonization by wild-type A. flavus was significantly reduced in treated plots compared with control plots, but total A. flavus/parasiticus colonization was not different between the two groups. A change to a more aggressive strain of A. flavus (NRRL 21882) as part of the biocontrol inoculum in 1997 resulted in a significantly (P < 0.001) higher colonization of corn by the applied strain. Weather conditions did not favor aflatoxin contamination in 1994 and 1995. In 1996, the aflatoxin concentration in corn from treated plots averaged 24.0 ppb, a reduction of 87% compared with the aflatoxin in control plots that averaged 188.4 ppb. In 1997, aflatoxin was reduced by 66% in treated corn (29.8 ppb) compared with control corn (87.5 ppb). Together, the data indicated that although the applied strain of A. parasiticus dominated in the soil, the nonaflatoxigenic strains of A. flavus were more responsible for the observed reductions in aflatoxin contamination. Inclusion of a nonaflatoxigenic strain of A. parasiticus in a biological control formulation for aflatoxin contamination may not be as important for airborne crops, such as corn, as for soilborne crops, such as peanuts.     

PCR-restriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize

Contamination of food and feedstuffs by Aspergillus species and their toxic metabolites is a serious problem as they have adverse effects on human and animal health. Hence, food contamination monitoring is an important activity, which gives information on the level and type of contamination. A PCR-based method of detection of Aspergillus species was developed in spiked samples of sterile maize flour. Gene-specific primers were designed to target aflR gene, and restriction fragment length polymorphism (RFLP) of the PCR product was done to differentiate Aspergillus flavus and Aspergillus parasiticus. Sterile maize flour was inoculated separately with A. flavus and A. parasiticus, each at several spore concentrations. Positive results were obtained only after 12-h incubation in enriched media, with extracts of maize inoculated with A. flavus (101 spores/g) and A. parasiticus (104 spores/g). PCR products were subjected to restriction endonuclease (HincII and PvuII) analysis to look for RFLPs. PCR-RFLP patterns obtained with these two enzymes showed enough differences to distinguish A. flavus and A. parasiticus. This approach of differentiating these two species would be simpler, less costly and quicker than conventional sequencing of PCR products.     

Distribution of aflatoxin-producing Aspergillus flavus and Aspergillus parasiticus in sugarcane fields in the southernmost islands of Japan

The distribution of Aspergillus flavus and Aspergillus parasiticus in sugarcane field soils and on harvested sugarcane stems was studied on seven islands of Okinawa and Kagoshima Prefectures, the southernmost prefectures in Japan. With the use of a combination of dilution plate and plant debris plate techniques, the fungi were detected on all seven islands studied and in 74% of 53 soil samples. The fungi were also found on the cut surfaces of sugarcane stems from one of the islands. A. parasiticus was the predominant fungus, although many atypical A. parasiticus isolates that produced metulated conidial heads were also obtained. The proportions of isolates testing positive for aflatoxin production were ca. 89% (146 of 164) of all isolates and ca. 69% of A. flavus isolates. More than 40% of A. flavus isolates also produced G aflatoxins. Scanning electron microscopic observation of conidial wall texture was useful in distinguishing A. parasiticus from A. flavus. Cyclopiazonic acid, an indole mycotoxin, was never synthesized by any of the A. parasiticus or G aflatoxin-producing A. flavus isolates tested.     

Studies on Aspergillus section Flavi isolated from maize in northern Italy

In 2003, for the first time in Italy, significant problems arose with colonization and contamination of maize destined for animal feed with Aspergillus section Flavi and aflatoxins (AFs). This resulted in milk and derived products being contaminated with AFM(1) at levels above the legislative limit. There was little knowledge and experience of this problem in Italy. The objectives of this research were thus to study the populations of Aspergillus section Flavi in six northern Italian regions and obtain information on the relative role of the key species, ability to produce sclerotia, production of the main toxic secondary metabolites, aflatoxins and cyclopiazonic acid, and tolerance of key environmental parameters. A total of 70 strains were isolated and they included the toxigenic species A. flavus and A. parasiticus. A. flavus was dominant in the populations studied, representing 93% of the strains. Seventy percent of strains of Aspergillus section Flavi produced AFs, with 50% of strains also producing cyclopiazonic acid. Sixty-two percent of A. flavus strains and 80% of A. parasiticus were able to produce sclerotia at 30 degrees C. Using 5/2 agar, only 1 strain developed S sclerotia and 19 L sclerotia. With regard to ecological studies, growth of Aspergillus section Flavi was optimal at between 25 and 30 degrees C, while AFB(1) production was optimal at 25 degrees C. Regarding water availability (water activity, a(w)), 0.99 a(w) was optimal for both growth and AFs production, while the only aflatoxin produced in the driest condition tested (0.83 a(w)) was AFB(1). This information will be very useful in identifying regions at risk in northern Italy by linking climatic regional information to levels of fungal contamination present and potential for aflatoxin production in maize destined for animal feed. This would be beneficial as part of a prevention strategy for minimising AFs in this product.     

Natural maize phenolic acids for control of aflatoxigenic fungi on maize

ABSTRACT:  Natural phytochemicals may be an alternative to synthetic chemicals for controlling fungal growth and mycotoxin production in stored maize. A key to progress in this field is to select the best natural maize phytochemicals to be applied in a storage maize ecosystem. This research was undertaken to evaluate the effects of the natural phytochemicals trans-cinnamic acid (CA) and ferulic acid (FA) alone at concentrations of 20 to 30 mM and in 5 combinations on Aspergillus flavus Link and A. parasiticus Speare populations and aflatoxin B₁ production. Studies on Aspergillus population and aflatoxin B₁ production were carried out in maize grain in relation to a water activity a_w of 0.99, 0.97, 0.95, and 0.93. CA and FA at concentrations of 25 to 30 mM, respectively, and CA-FA mixture T9 (25 + 30 mM) were the treatments most effective at inhibiting A. flavus and A. parasiticus population at all a_w assayed after 11 d of incubation. At all a_w values, the mixture CA-FA T9 (25 + 30 mM) completely inhibited (100%) aflatoxin B₁ production by both strains at a_w= 0.99, 0.97, 0.95, and 0.93. Decreased aflatoxin B₁ levels in comparison with the control were observed with mixtures CA-FA T6 (10 + 25 mM), T7 (20 + 20 mM), and T8 (20 + 30 mM) of both strains in the majority of a_w assayed. The data show that CA and FA could be considered as effective fungitoxicants for A. flavus and A. parasiticus in maize in the a_w range 0.99 to 0.93. The information obtained shows promise for controlling aflatoxigenic fungi in stored maize.     

Comparison of Four Media for the Enumeration of Fungi in Dairy Products — A Collaborative Study

Four fungal enumeration media, including potato dextrose agar (PDA) with antibiotics (APDA), acidified PDA (PDA-A), Cooke rose bengal agar (CRBA), and APDA containing the mold colony spreading inhibitor dichloran (APDA-D-5), were compared by seven laboratories for recovery of yeasts and molds from inoculated and naturally contaminated dairy products. Although some variability was observed in counts obtained for individual products between laboratories, no significant overall differences were observed among the four media. Mold colony diameters but not numbers were reduced with APDA-D-5 and CRBA, thus aiding in the counting of plates heavily contaminated with molds.     

Mold counts and Aspergillus section Flavi populations in rice and its by-products from the Philippines

Mold counts and Aspergillus section Flavi populations in rice and its by-products from the Philippines were examined. The average mold counts of rough rice, brown rice, and locally produced polished rice were 4.1 x 10(3), 1.0 x 10(3), and 1.1 x 10(3) CFU/g, respectively. Average Aspergillus section Flavi counts of the same samples were 3.0 x 10(2), 1.1 x 10(2), and 2.6 x 10(2) CFU/g, respectively. Twenty-seven percent of mold isolates from rough rice, polished rice, and brown rice were section Flavi spp., 31% of which were toxigenic. No section Flavi isolates were obtained from imported rice samples from Thailand and Vietnam. Aspergillus section Flavi was also isolated from rice hull, rice bran, and settled dust from rice milling operations. Toxigenic isolates of both Aspergillus flavus and Aspergillus parasiticus were present in at least one sample of each type of rice and rice by-product except settled dust. Aflatoxins produced in vitro by the isolates ranged from <1 microg/kg to 6,227 microg/kg. A. flavus isolates produced only B aflatoxins, whereas A. parasiticus isolates produced both B and G aflatoxins. Although total mold counts of Philippine rice and its by-products are within tolerable limits, the establishment of maximum limits in counts of potentially aflatoxigenic species in foods and feeds is important because the mere presence of toxin producers is considered a possible risk factor. The results of this research illustrate the need for strict monitoring of rice during both storage and marketing, especially in warm and humid seasons when infestation and consequent production of aflatoxins by Aspergillus section Flavi is expected.     

Comparison of a simple butterfat agar medium with other media used for isolation and enumeration of lipolytic bacteria from dairy products

A nutrient agar medium containing 0.1% of a low melting point fraction of butterfat was shown to be suitable for detection, enumeration and isolation of lipolytic bacteria from milk. Bacterial growth was not inhibited by the butterfat and lipolytic reactions were clearly visible and easily interpreted. Lipolytic counts on the butterfat agar compared favourably with lipolytic counts obtained with other commonly used media.