Microbiological quality, fungal diversity and aflatoxins contamination in carob flour (Prosopis flexuosa).

Carob flour is obtained from pods of some species of Prosopis, leguminous trees that abound in many desert habitats worldwide. Currently, this product is available in healthy food stores in several countries, including Argentina, as a nontraditional meal of growing interest with multiple applications for the preparation of puddings, biscuits and snacks, among others. The objective of the present study was to evaluate the microbial quality of carob flour on basis of the presence of deteriorative and pathogenic microorganisms. Fungal diversity of the mycobiota was also studied with a special interest in toxigenic fungi. Eighteen samples of carob flour (Prosopis flexuosa) were analysed. Standard plate count of aerobic mesophilic bacteria showed levels of contamination ranging from <102 (estimative) to 6.8 × 105 CFU/g; total coliforms from <102 (estimative) to 4.7 × 105 CFU/g; moulds and yeasts from 2.1 × 102 to 8.1 × 104. In all samples, the absence of Salmonella sp. was verified in 25 g and counts of Bacillus cereus less than 102 were observed. These results indicate that from the safety point of view the carob flour studied does not have a significant microbial load. Regarding to fungal contamination, Aspergillus and Penicillium were the genera more diverse in species and were present in all the samples. Some of the species identified were potential mycotoxins producers. Among the most frequently detected species in the studied mycobiota were the Aspergillus of the Flavi section, well recognized as potential aflatoxin producers. The A. flavus species was one of the most widely distributed, since it was detected in almost all samples. A. parasiticus and A. arachidicola were found more sporadically. Aflatoxins analysis demonstrated that a high proportion of the samples were contaminated with aflatoxins in concentrations relatively low, ranging from 1.26 to 20.33 µg/kg of total aflatoxins. Type G aflatoxins are much less frequent contaminants than type B aflatoxins, which is consistent with the fact that A. parasiticus and A. arachidicola (producers of type B and G aflatoxins) were detected sporadically, while A. flavus, which produces aflatoxins B1 and B2, was present in a high number of samples. Results of the present work indicate that carob flour is susceptible to Aspergillus section Flavi and aflatoxin contamination and should be subjected to aflatoxin monitoring prior to marketing as required for other traditional crops.

Microbiological quality of Argentinian paprika / Calidad microbiológica del pimentón argentino

The aim of this study was to evaluate the microbiological quality of paprika produced in Catamarca, Argentina. Microbiological analyses were carried out for the enumeration of total aerobic mesophilic bacteria, coliforms, yeasts and molds, and the detection of Salmonella in samples obtained from different local producers during three consecutive years. The mycobiota was identified paying special attention to the mycotoxigenic molds. Standard plate counts of aerobic mesophilic bacteria ranged from 2.7 x 10(5) to 3.7 x 10(7)CFU/g. Coliform counts ranged from <10 to 8.1 x 10(4) CFU/g. Salmonella was not detected in any of the samples tested. Fungal counts (including yeasts and molds) ranged between 2 x 10² and 1.9 x 10(5) CFU/g. These results showed a high level of microbial contamination, exceeding in several samples the maximum limits set in international food regulations. The study of the mycobiota demonstrated that Aspergillus was the predominant genus and Aspergillus niger (potential producer of ochratoxin A) the most frequently isolated species, followed by Aspergillus flavus (potential producer of aflatoxins). Other species of potential toxigenic fungi such as Aspergillus ochraceus, Aspergillus westerdijkiae, Penicillium chrysogenum, Penicillium crustosum, Penicillium commune, Penicillium expansum and Alternaria tenuissima species group were encountered as part of the mycobiota of the paprika samples indicating a risk of mycotoxin contamination. A. westerdijkiae was isolated for the first time in Argentina.

El pimentón es considerado una de las especias más proclives a contaminarse con diversos tipos de microorganismos, incluyendo patógenos como Salmonella y hongos capaces de producir micotoxinas. Existen muy pocos datos acerca de la contaminación microbiana del pimentón producido en nuestro país. El objetivo del presente trabajo fue evaluar la calidad microbiológica del pimentón (Capsicum annum L.) producido en la provincia de Catamarca, una de las principales zonas productoras del norte argentino. Se realizó el recuento de bacterias aerobias mesófilas, coliformes totales y mohos y levaduras, y la búsqueda de Salmonella en muestras obtenidas de diferentes establecimientos productores locales durante 3 años consecutivos. Se identificaron todas las cepas fúngicas (1.622 aislamientos) a nivel de género y se determinaron las especies pertenecientes a los géneros potencialmente toxinógenos. Los recuentos totales de bacterias aerobias mesófilas variaron entre 2,7 x 10(5)y3,7 x 10(7)UFC/g. Los coliformes totales estuvieron en el rango de < 10 a 8,1 x 10(4) UFC/g. Salmonella no fue detectada en ninguna de las muestras analizadas. Los resultados obtenidos muestran un alto nivel de contaminación, que excede en varias de las muestras los límites máximos establecidos en las regulaciones alimentarias internacionales. El estudio de la micobiota demostró que Aspergillus fue el género predominante. Otros géneros encontrados fueron Cladosporium, Rhizopus, Alternaría y Penicillium. Aspergillus niger (potencial productor de ocratoxina A) fue la especie aislada con mayor frecuencia, seguida de Aspergillus flavus (potencial productor de aflatoxinas). También se encontraron otras especies toxinógenas, lo que indica un riesgo potencial de contaminación con micotoxinas. Aspergillus westerdijkiae fue aislado por primera vez en Argentina.

Microbiological quality of Argentinian paprika.

The aim of this study was to evaluate the microbiological quality of paprika produced in Catamarca, Argentina. Microbiological analyses were carried out for the enumeration of total aerobic mesophilic bacteria, coliforms, yeasts and molds, and the detection of Salmonella in samples obtained from different local producers during three consecutive years. The mycobiota was identified paying special attention to the mycotoxigenic molds. Standard plate counts of aerobic mesophilic bacteria ranged from 2.7×105 to 3.7×107CFU/g. Coliform counts ranged from <10 to 8.1×104CFU/g. Salmonella was not detected in any of the samples tested. Fungal counts (including yeasts and molds) ranged between 2×102 and 1.9×105CFU/g. These results showed a high level of microbial contamination, exceeding in several samples the maximum limits set in international food regulations. The study of the mycobiota demonstrated that Aspergillus was the predominant genus and Aspergillus niger (potential producer of ochratoxin A) the most frequently isolated species, followed by Aspergillus flavus (potential producer of aflatoxins). Other species of potential toxigenic fungi such as Aspergillus ochraceus, Aspergillus westerdijkiae, Penicillium chrysogenum, Penicillium crustosum, Penicillium commune, Penicillium expansum and Alternaria tenuissima species group were encountered as part of the mycobiota of the paprika samples indicating a risk of mycotoxin contamination. A. westerdijkiae was isolated for the first time in Argentina.

Abiotic factors and their interactions influence on the co-production of aflatoxin B(1) and cyclopiazonic acid by Aspergillus flavus isolated from corn.

The objectives of this study were i) to determine the effects of the interactions of water activity, temperature and incubation time on the co-production of AFB1 and CPA by isolates of Aspergillus flavus with different profile of mycotoxin production and ii) to identify the aW and temperature limiting conditions for the production of both mycotoxins. Fungi used in this study were selected because they belonged to different chemotypes: chemotype I (AFB1+/CPA+), III (AFB1+/CPA-) and IV (AFB1-/CPA+), respectively. Two culture media were used; Czapek yeast agar (CYA) and corn extract agar (CEM), at different incubated temperatures (10-40 °C) and aW levels (0.80-0.98). AFB1 and CPA production were analyzed after 7, 14, 21 and 28 days of incubation. Significant differences were observed with respect to mycotoxin production depending on the media evaluated. The AFB1 production occurred more favorably on CYA while the highest CPA concentrations were recorded on CEM. Within the range of aW evaluated in this study, 0.83 was the limiting level for both toxins production. The optimum conditions for AFB1 production occurred at 0.96 aW and 30 °C after 21 days of incubation, regardless of the media and isolate. Although different amounts of toxins were produced in each medium, the limiting and optimum conditions for their production were similar in both. No differences in the response of the three isolates to the abiotic factors discussed were observed despite belonging to different chemotypes. The determination of the thresholds of mycotoxins co-production, especially in the case of data obtained with the corn extract medium can be useful to avoid the conditions conducive to co-occurrence of these mycotoxins in corn.

Ochratoxin A production by a mixed inoculum of Aspergillus carbonarius at different conditions of water activity and temperature.

Growth rate, lag phase and OTA production of a mixed inoculum of four Aspergillus carbonarius strains were compared to the behaviour of the single strains at 30 degrees C on Czapek Yeast Extract Agar. Significant differences between radial growth rates and lag phases of the different isolates were observed; however, no significant differences between growth rates of each strain and the mixed inoculum were detected. When the four strains were inoculated simultaneously, the lag phase was the same than the higher value obtained for individual strains, suggesting mycelial interactions between the A. carbonarius isolates. The four strains differed in maximum OTA yield, and the toxin accumulation by the mixed inoculum showed intermediate levels at each time point. The effects of water activity (0.83, 0.85, 0.87, 0.89, 0.90, 0.93 and 0.95) and temperature (15, 20, 25, 30, and 35 degrees C) on OTA production by the mixed inoculum were studied at 7, 14, 21 and 28days of incubation. The limiting water activity for OTA production by the mixed inoculum was 0.87, showing xerotolerant behaviour of the strains isolated from dried vine fruits. Results obtained were similar to those reported for single A. carbonarius strains from European countries, Israel, Australia and South America. The similar trend in the response of the different isolates to the variation of environmental parameters may be of interest for the building of predictive models.

Inhibition of growth and ochratoxin A biosynthesis in Aspergillus carbonarius by flavonoid and nonflavonoid compounds.

The effect of naturally occurring phenolic compounds on Aspergillus carbonarius growth and ochratoxin A (OTA) production was studied. Caffeic acid and the flavonoids, rutin and quercetin, were added to Czapek Yeast Extract agar at concentrations ranging between 50 and 500 mg/l. All phenolic compounds had a significant influence on growth rate and lag phase of A. carbonarius at 250 mg/l. The growth was completely inhibited with 500 mg/l. In comparison with the control, a significant decrease in OTA production was observed with all phenolic compounds. In general, effect on growth was less evident than effect on toxin production. An inhibitory effect on growth and OTA production, as concentration was increased was observed in all cases. The response of A. carbonarius to the flavonoids, rutin and quercetin, was similar. The inhibitory effect of these natural phenolic compounds on fungal growth and OTA production could be an alternative to the use of chemical fungicides.

Effect of processing for saponin removal on fungal contamination of quinoa seeds (Chenopodium quinoa Willd.).

Incidence of fungal contamination of quinoa seeds from three locations (Salar de Uyuni, Bolivia; Salta and Tucumán provinces, Argentina) was analyzed in samples with and without treatment to remove saponins (wet method). In processed samples, the percentage of infection was reduced. Distribution of the different fungal genera was not homogeneous in the three locations (p<0.05), although Penicillium and Aspergillus were the most prevalent contaminants, regardless the geographic origin of the samples. Other genera, such as Eurotium, Fusarium, Phoma, Ulocladium, Mucor and Rhizopus were less frequently isolated. Absidia, Alternaria, Cladosporium, Dreschlera, Epicoccum and Monascus were sporadically encountered. Significant differences (p<0.05) in the distribution of fungal genera in samples with and without saponins from each location were observed. In all cases, processing caused a decrease of Aspergillus incidence, while increased the proportion of Penicillium, Eurotium, Mucor and Rhizopus indicating that these genera were part of the internal mycota. A. flavus and A. niger were the dominating species of genus Aspergillus. A similar pattern of prevalent Penicillium species was observed in samples with and without saponins, since P. aurantiogriseum, P.chrysogenum, P. citrinum and P. crustosum were always present in high number, although their relative density was variable according to the geographic origin of samples. Mycotoxin-producing ability of most representative species was also determined. Toxigenic strains of A. flavus (aflatoxins and cyclopiazonic acid), A. parasiticus (aflatoxins), P. citrinum (citrinin) and P. griseofulvum (cyclopiazonic acid) were found. None of the A. niger isolates was ochratoxin A producer. The above mentioned mycotoxins were not detected in the samples analyzed.

Two novel aflatoxin-producing Aspergillus species from Argentinean peanuts.

Two novel species from Aspergillus section Flavi from different species of Arachis (peanuts) in Argentina are described as Aspergillus arachidicola sp. nov. and Aspergillus minisclerotigenes sp. nov. Their novel taxonomic status was determined using a polyphasic taxonomic approach with phenotypic (morphology and extrolite profiles) and molecular (beta-tubulin and calmodulin gene sequences) characters. A. minisclerotigenes resembles Aspergillus flavus and Aspergillus parvisclerotigenus in producing aflatoxins B(1) and B(2), cyclopiazonic acid, kojic acid and aspergillic acid, but in addition it produces aflatoxins G(1) and G(2), aflavarins, aflatrem, aflavinines, parasiticolides and paspaline. This species also includes several isolates previously assigned to A. flavus group II and three Australian soil isolates. A. arachidicola produces aflatoxins B(1), B(2), G(1) and G(2), kojic acid, chrysogine and parasiticolide, and some strains produce aspergillic acid. The type strain of A. arachidicola is CBS 117610(T) =IBT 25020(T) and that of A. minisclerotigenes is CBS 117635(T) =IBT 27196(T). The Mycobank accession numbers for Aspergillus minisclerotigenes sp. nov. and Aspergillus arachidicola sp. nov. are respectively MB 505188 and MB 505189 (http://www.mycobank.org).

[Putative mycotoxin-producing fungi isolated from alpataco (Prosopis flexuosa) fruits]. / Hongos toxicogénicos contaminantes de frutos de alpataco.

Fungi contaminant of alpataco (Prosopis flexuosa) fruits from La Pampa province (Argentina) were identified. Alternaria alternata and Sphaeropsis sapinea were the dominant species. Phoma sp., Nigrospora sp., Preussia minima, Cladosporium sp., Pithomyces chartarum, Epicoccum nigrum, Aspergillus niger and Aspergillus speluneus were also isolated but with less frequency. Twelve strains of Alternaria alternata, the toxigenic species with higher incidence, were screened for alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) production. Since one isolate was able to produce AME, six isolates produced AOH and AME and two isolates produced AOH, AME and TA, these results indicate a potential risk of contamination with Alternaria toxins in this substrate.

Hongos toxicogénicos contaminantes de frutos de alpataco / Putative mycotoxin-producing fungi isolated from “alpataco” (Prosopis flexuosa) fruits

Se estudió la presencia de hongos potencialmente toxicogénicos en frutos dealpataco (Prosopis flexuosa D.C. var. depressa Roig) recolectados en laprovincia de La Pampa, Argentina. Las especies predominantes fueronAlternaria alternata y Sphaeropsis sapinea. En menor proporción se aislaronPhoma sp., Nigrospora sp., Preussia minima, Cladosporium sp., Pithomyceschartarum, Epicoccum nigrum, Aspergillus niger y Aspergillus speluneus.La capacidad para producir micotoxinas se determinó en 12 cepas deAlternaria alternata, única especie potencialmente toxicogénica aislada conrelativa frecuencia. Dos cepas produjeron ácido tenuazónico (AT), alternariol(AOH) y alternariol-metil-éter (AME), seis produjeron AOH y AME, una produjosolamente AME y las tres restantes resultaron no toxicogénicas. Losresultados de este estudio preliminar indican un riesgo potencial decontaminación con toxinas de Alternaria en la harina de alpataco, de crecienteuso en la alimentación humana y animal en ciertas áreas geográficas(AU)

Fungi contaminant of “alpataco” (Prosopis flexuosa) fruits from “La Pampaprovince” (Argentina) were identified. Alternaria alternata and Sphaeropsissapinea were the dominant species. Phoma sp., Nigrospora sp., Preussiaminima, Cladosporium sp., Pithomyces chartarum, Epicoccum nigrum,Aspergillus niger and Aspergillus speluneus were also isolated but with lessfrequency. Twelve strains of Alternaria alternata, the toxigenic species withhigher incidence, were screened for alternariol (AOH), alternariol monomethylether (AME) and tenuazonic acid (TA) production. Since one isolate was ableto produce AME, six isolates produced AOH and AME and two isolatesproduced AOH, AME and TA, these results indicate a potential risk ofcontamination with Alternaria toxins in this substrate(AU)

Analysis of population structure of Aspergillus flavus from peanut based on vegetative compatibility, geographic origin, mycotoxin and sclerotia production.

Isolates of Aspergillus flavus obtained from a new growing peanut region in Argentina (Formosa province) were examined for aflatoxin types B and G and cyclopiazonic acid (CPA) production. Sclerotia diameters and the number of sclerotia produced per square centimetre were also determined for each isolate. They were tested by vegetative compatibility group analysis to investigate their genetic relatedness and correlate the results with vegetative compatibility groups previously described from the major peanut-growing area (Córdoba province) in our country. Two isolates were considered atypical because they simultaneously produce aflatoxins B and G and CPA. A. flavus population from Formosa province was very diverse genetically. Vegetative compatibility groups (VCGs) formed by typical isolations of A. flavus were different among agroecological sites. Formosa isolates could not be grouped to any of the Córdoba VCGs, while that one of the VCGs that contain atypical isolates included strains from the two geographical regions. Each VCG included isolates of the same mycotoxin and sclerotia production pattern. The two regions analysed have different climatic conditions, soil type, crop sequence history and also are in different latitude. These parameters may reflect different geographic adaptation between isolates from both sites.

Variability of aflatoxin and cyclopiazonic acid production by Aspergillus section flavi from different substrates in Argentina.

Aspergillus section flavi strains isolated from peanuts, wheat and soybean grown in Argentina were screened for aflatoxins (type B and G) and cyclopiazonic acid (CPA) production. Aspergillus flavus was the predominant species in all substrates, although there was almost the same proportion of A. flavus and Aspergillus parasiticus in peanuts. Aspergillus nomius was not found. Incidence of aflatoxigenic A. flavus strains was higher in peanuts (69%) than in wheat (13%) or soybeans (5%) while the ratio of CPA producers A. flavus isolated from all substrates was very high (94% in peanuts, 93% in wheat and 73% in soybeans). Isolates of A. flavus able to produce simultaneously aflatoxins type B and CPA were detected in all substrates, suggesting the possibility of co-occurrence of these toxins. Almost all isolates of A. parasiticus resulted aflatoxins (type B and G) producers but did not produce CPA. Five of sixty-seven strains isolated from peanuts showed an unusual pattern of mycotoxin production (aflatoxins type B and G simultaneously with CPA). These strains also produced numerous small sclerotia like S strains of A. flavus detected in cottonseed in Arizona and in soils of Thailand and West Africa. The atypical strains are not widely distributed in Argentina and were found uniquely in peanuts.

Aflatoxin Production in Soybean Varieties Grown in Argentina.

Natural occurrence of aflatoxin contamination on soybeans was investigated. Thirty four samples from the 1986 crop (5.8% positive samples) and 60 samples harvested in 1987 (11.6% positive samples) were analyzed. Aflatoxin levels in positive samples were low, ranging from traces to 36 µg/kg of total aflatoxins. Aflatoxin production by three isolates of Aspergillus flavus / A. parasiticus in 13 soybean varieties has been studied. Soybeans used for cultures were surface disinfected. Levels of aflatoxin formed were dependent both on the toxicogenic potential of the fungal isolate and on the variety of soybeans. Under laboratory conditions variety Prata was the most susceptible and variety SRF the less susceptible to aflatoxin formation.