Experimental aflatoxin production in Manchego-type cheese

Manchego-type cheese, a typical Spanish cheese, was inoculated in various ways with an aflatoxigenic organism, Aspergillus parasiticus NRRL 2999, to study the production of aflatoxin. When the original milk was contaminated with a spore suspension, aflatoxin was not detected in paraffin-covered cheeses although it was present in the top layer of non-paraffin-covered cheeses after ripening at 15°C for 60 d. When the cheese surface was inoculated, no aflatoxins were detected in paraffin-covered cheeses after ripening for 60 d although they were found when the cheeses were ripened for 30 d. In non-paraffin-covered cheeses aflatoxins were detected only in the top layer and in the second 10 mm layer when cheeses were incubated after the normal ripening at 28°C for 30 d. When the centre of the cheese was inoculated, no aflatoxins were detected although Aspergillus grew slightly along the inoculation area. When cheese portions were inoculated, fungal growth was evident after incubation at 28° and 15°C for 6 d but there was no growth at 10°C after 50 d. At 28°C aflatoxins were detected at a concentration of 132 μg/g after 13 d, the highest level obtained. In cheese paste at 28° and 15°C, growth was intense, but the level of aflatoxins detected was lower than in cheese portions. At 10°C the growth was heavy, but aflatoxins were not detected.     

Influence of white light on production of aflatoxins and anthraquinones in Aspergillus parasiticus.

The effect of continuous light and continuous darkness on the growth of Aspergillus parasiticus and on the production of aflatoxin, averufin, versicolorin A, and versicolorin C by Aspergillus parasiticus were determined at six different temperatures with six replicates for each experiment. No growth was observed at 15 degrees C in the light, although slight growth was observed at this temperature in the dark. No aflatoxins or anthraquinones were produced in the light or dark at 35 and 40 degrees C, although growth was good at these temperatures. Differences in aflatoxins and anthraquinones for cultures grown in light and in dark were consistent at each temperature. Higher mean quantities of these secondary metabolites were produced in the light at 20 and 25 degrees C; lower mean quantities were produced in the light at 30 degrees C. The ranges of values overlapped considerably, but in all cases the differences between temperatures were significant.     

Experimental short time production of aflatoxins by Aspergillus parasiticus in mixed feeds as related to various moisture contents

Experimental short time production of aflatoxins in mixed feeds at 22, 28 and 37 °C as related to various moisture contents was studied. Growth of Aspergillus parasiticus was not observed in the meals with a moisture content ranging around 15% (22, 28 and 37 °C); the lowest quantifiable total aflatoxins at the fourth day was detected at 22 °C with 19.4% of moisture content; the higher total quantity of aflatoxins (113 mg/kg) was produced at 28 °C with 29.3% of moisture content. The ratio aflatoxin B₁/aflatoxin G₁ increased as the temperature raised.     

Aflatoxins in sunflower seeds: Influence of Alternaria alternata on aflatoxin production by Aspergillus parasiticus

The aim of the present work was to determine the influence of Alternaria alternata upon aflatoxin production by Aspergillus parasiticus.A mixture of spores of both strains was inoculated in sunflower seeds at 0,90 a_w, and incubated for 42 days at 28 °C ±1.The cultures were observed and analyzed every 7 days to determine the infection level of the seeds and the production of aflatoxins. Results showed that when the seeds were inoculated only with Aspergillus parasiticus, 100% were infected from the 7th day.When Aspergillus parasiticus and Alternaria alternata were simultaneously inoculated the infection level of the seeds was 100% for Aspergillus parasiticus following 7 days of inoculation and 0% for Alternaria alternata. After the 14th day of inoculation there was no significant difference in the infection percentage of both strains (approximately 80% of each one). As far as toxin production is concerned a remarkable decrease was observed when seeds were inoculated with both strains simultaneously.In accordance to the results, Alternaria alternata would not compete with Aspergillus parasiticus in colonization of seeds but would either degrade the aflatoxins by Aspergillus parasiticus or compete for aflatoxin biosynthesis precursors. Alternaria alternata could also secrete some substance that specifically inhibits aflatoxin synthesis.     

Simple fluorescence method for rapid estimation of aflatoxin levels in a solid culture medium

Aflatoxin concentrations in agar media were estimated with a direct technique that quantifies the fluorescence of agar containing aflatoxins. Tubes containing 5 ml of an agar medium inoculated with spores of aflatoxin-producing Aspergillus isolates were incubated for 3 days at 30 degrees C and set in a carriage specifically designed to carry culture tubes in a scanning densitometer. Fluorescence (450 nm and above) was elicited in the agar by UV light (365 nm) and photometrically measured. Agar fluorescence directly correlated (r2 = 0.89 +/- 0.05, P less than 0.001) with the concentration of aflatoxin within the range 0 to 18.7 micrograms/g. The lowest aflatoxin concentration detected was 50 ng/g. The technique successfully differentiated the aflatoxigenic potentials of Aspergillus isolates.     

Simple fluorescence method for rapid estimation of aflatoxin levels in a solid culture medium.

Aflatoxin concentrations in agar media were estimated with a direct technique that quantifies the fluorescence of agar containing aflatoxins. Tubes containing 5 ml of an agar medium inoculated with spores of aflatoxin-producing Aspergillus isolates were incubated for 3 days at 30 degrees C and set in a carriage specifically designed to carry culture tubes in a scanning densitometer. Fluorescence (450 nm and above) was elicited in the agar by UV light (365 nm) and photometrically measured. Agar fluorescence directly correlated (r2 = 0.89 +/- 0.05, P less than 0.001) with the concentration of aflatoxin within the range 0 to 18.7 micrograms/g. The lowest aflatoxin concentration detected was 50 ng/g. The technique successfully differentiated the aflatoxigenic potentials of Aspergillus isolates.     

Intrafungal distribution of aflatoxins among conidia and sclerotia of Aspergillus flavus and Aspergillus parasiticus

This research examines the distribution of aflatoxins among conidia and sclerotia of toxigenic strains of Aspergillus flavus Link and Aspergillus parasiticus Speare cultured on Czapek agar (21 days, 28 degrees C). Total aflatoxin levels in conidia and sclerotia varied considerably both within (intrafungal) and among strains. Aspergillus flavus NRRL 6554 accumulated the highest levels of aflatoxin (conidia: B1, 84000 ppb; G1, 566000 ppb; sclerotia: B1, 135000 ppb; G1, 968000 ppb). Substantial aflatoxin levels in conidia could place at risk those agricultural workers exposed to dust containing large numbers of A. flavus conidia. Cellular ratios of aflatoxin B1 to aflatoxin G1 were nearly identical in conidia and sclerotia even though levels of total aflatoxins in these propagule types may have differed greatly. Aflatoxin G1 was detected in sclerotia of all A. flavus strains but in the conidia of only one strain. Each of the A. parasiticus strains examined accumulated aflatoxin G1 in both sclerotia and conidia. These results are examined in the context of current evolutionary theory predicting an increase in the chemical defense systems of fungal sclerotia, propagules critical to the survival of these organisms.     

Biosynthetic relationship among aflatoxins B1, B2, M1, and M2.

Aflatoxins are a family of toxic, acetate-derived decaketides that arise biosynthetically through polyhydroxyanthraquinone intermediates. Most studies have assumed that aflatoxin B1 is the biosynthetic precursor of the other aflatoxins. We used a strain of Aspergillus flavus which accumulates aflatoxin B2 to investigate the later stages of aflatoxin biosynthesis. This strain produced aflatoxins B2 and M2 but no detectable aflatoxin B1 when grown over 12 days in a low-salt, defined growth medium containing asparagine. Addition of dichlorvos to this growth medium inhibited aflatoxin production with concomitant accumulation of versiconal hemiacetal acetate. When mycelial pellets were grown for 24, 48, and 72 h in growth medium and then transferred to a replacement medium, only aflatoxin B2 and M2 were recovered after 96 h of incubation. Addition of sterigmatocystin to the replacement medium led to the recovery of higher levels of aflatoxins B2 and M2 than were detected in control cultures, as well as to the formation of aflatoxins B1 and M1 and O-methylsterigmatocystin. These results support the hypothesis that aflatoxins B1 and B2 can arise independently via a branched pathway.     

Biosynthetic relationship among aflatoxins B1, B2, M1, and M2

Aflatoxins are a family of toxic, acetate-derived decaketides that arise biosynthetically through polyhydroxyanthraquinone intermediates. Most studies have assumed that aflatoxin B1 is the biosynthetic precursor of the other aflatoxins. We used a strain of Aspergillus flavus which accumulates aflatoxin B2 to investigate the later stages of aflatoxin biosynthesis. This strain produced aflatoxins B2 and M2 but no detectable aflatoxin B1 when grown over 12 days in a low-salt, defined growth medium containing asparagine. Addition of dichlorvos to this growth medium inhibited aflatoxin production with concomitant accumulation of versiconal hemiacetal acetate. When mycelial pellets were grown for 24, 48, and 72 h in growth medium and then transferred to a replacement medium, only aflatoxin B2 and M2 were recovered after 96 h of incubation. Addition of sterigmatocystin to the replacement medium led to the recovery of higher levels of aflatoxins B2 and M2 than were detected in control cultures, as well as to the formation of aflatoxins B1 and M1 and O-methylsterigmatocystin. These results support the hypothesis that aflatoxins B1 and B2 can arise independently via a branched pathway.     

Influence of temperature cycling on the production of aflatoxins B1 and G1 by Aspergillus parasiticus.

The effect of temperature cycling on the relative productions of aflatoxins B1 and G1 by Aspergillus parasiticus NRRL 2999 was studied. The cycling of temperature between 33 and 15 degrees C favored aflatoxin B1 accumulation, whereas cycling between 35 and 15 degrees C favored aflatoxin G1 production. Cultures subjected to temperature cycling between 33 and 25 degrees C at various time intervals changed the relative productions of aflatoxins B1 and G1 drastically. Results obtained with temperature cycling and yeast extract-sucrose medium with ethoxyquin to decrease aflatoxin G1 production suggest that the enzyme system responsible for the conversion of aflatoxin B1 to G1 might be more efficient at 25 degrees C than at 33 degrees C. The possible explanation of the effect of both constant and cycling temperatures on the relative accumulations of aflatoxins B1 and G2 might be through the control of the above enzyme system. The study also showed that greater than 57% of aflatoxin B1, greater than 47% of aflatoxin G1, and greater than 50% of total aflatoxins (B1 plus G1) were in the mycelium by day 10 under both constant and cyclic temperature conditions.     

Survival of surface ripening cultures during storage and monitoring their development on cheese

AIMS: To study the survival of bacteria isolated from the surface of smear cheese and monitor their development during cheese ripening. METHODS AND RESULTS: The storage of five potential bacterial surface-ripening cheese cultures, Brevibacterium aurantiacum, Corynebacterium casei, Corynebacterium variable, Microbacterium gubbeenense and Staphylococcus saprophyticus, in maximum recovery diluent (MRD), containing 0.85% w/v or 5% w/v NaCl, at 21 or 4 degrees C for 40 days, was investigated. All five strains studied survived well with a maximum decrease of c. 2.5 log(10) CFU ml(-1) after storage for 40 days at 4 degrees C in 0.85% or 5% w/v NaCl. Survival, especially of C. variable, was less at 21 degrees C. The development of defined ripening cultures containing C. casei and Debaryomyces hansenii on two farmhouse cheeses was also evaluated. Using pulsed-field gel electrophoresis (PFGE) for the bacteria and mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) for the yeast, it was shown that the ripening cultures could be re-isolated in high numbers, 10(8) CFU cm(-2) for C. casei and 10(6) CFU cm(-2) for D. hansenii, from the cheese surface after 2.5 weeks of ripening. CONCLUSIONS: Ripening strains of surface ripening cultures can be stored in MRD containing 5% w/v salt at 4 degrees C for at least 40 days. Such cultures are recovered in high numbers from the cheese during ripening. SIGNIFICANCE AND IMPACT OF STUDY: This study has provided a low-cost and efficient way to store bacteria that could be used as ripening cultures for smear cheese. Such cultures can be recovered in high numbers from the cheese surface during ripening.     

Increased production of aflatoxins by Aspergillus parasiticus Speare in the presence of rubratoxin B.

The influence of rubratoxin B, a metabolite of Penicillium rubrum Stoll, on the growth and aflatoxin production of a strain of Aspergillus parasiticus Speare grown in the chemically defined medium of Reddy et al. (Appl. Microbiol. 22:393-396, 1971) was studied. After 4 days of incubation on a rotary shaker at 25 degrees C, the presence of 10 microgram/ml caused 45 to 50% reduction in dry weight production, although at the same concentration of rubratoxin B, the reduction of growth after 10 days was only 15%. In the presence of 50 microgram/ml there was a reduction in dry weight production of 94% after 4 days of incubation, and it was still 86% after 8 days. Rubratoxin B concentrations of 50 microgram/ml and higher usually caused a reduction in aflatoxin production in the medium comparable with the reduction in biomass, but at concentrations as low as 10 microgram/ml, there was a pronounced increase in the production of aflatoxins, especially of G1, despite the reduction in biomass. The ecological significance of these observations is discussed.     

Microbiological profile in Serra ewes' cheese during ripening

The microflora of Serra cheese was monitored during a 35 d ripening period at three different periods within the ewe's lactation season. After 7 d ripening, the numbers of micro-organisms reached their maximum, and lactic acid bacteria (LAB) and coliforms were the predominant groups. Pseudomonads were not detected after 1 week of ripening. At all stages of ripening, cheeses manufactured in spring exhibited the lowest numbers of LAB and yeasts, whereas cheeses manufactured in winter showed the lowest numbers of coliforms and staphylococci.
Leuconostoc lactis was the most abundant LAB found in Serra cheese whereas Enterococcus faecium and Lactococcus lactis spp. lactis exhibited the highest decrease in percentage composition. Numbers of both Leuc. mesenteroides and Lactobacillus paracasei tended to increase throughout ripening. The most abundant coliform was Hafnia alvei. Klebsiella oxytoca was found in curd but declined in number during ripening. Staphylococcal flora of curd was mainly composed of Staphylococcus xylosus, Staph. aureus and Staph. epidermidis. Staphylococcus xylosus was the major species found at the end of ripening. Pseudomonas fluorescens , was the only Pseudomonas species isolated from the curd. Although a broad spectrum of yeasts were found in Serra cheese, Sporobolomyces roseus was the most abundant yeast isolated.     

Aflatoxin Production in Meats. I. Stored Meats

Aflatoxins were produced on fresh beef (in which bacterial spoilage was delayed with antibiotics), ham, and bacon inoculated with toxinogenic fungi and stored at 15, 20 and 30 C. Meats stored at 10 C were spoiled by bacteria and yeast before detectable levels of aflatoxins were produced. High levels of aflatoxins were formed in meats stored at 20 C; one sample supported the production of 630 mug of aflatoxins per g of meat, the major portion (580 mug) of which was aflatoxin G(1). Meats stored below 30 C developed higher levels of aflatoxin G(1) than B(1), but at 30 C Aspergillus flavus produced equal amounts of B(1) and G(1), whereas A. parasiticus continued to produce more G(1) than B(1).     

Aflatoxin production by entomopathogenic isolates of Aspergillus parasiticus and Aspergillus flavus

Fourteen isolates of Aspergillus parasiticus and 2 isolates of Aspergillus flavus isolated from the mealybug Saccharicoccus sacchari were analyzed for production of aflatoxins B1, B2, G1, and G2 in liquid culture over a 20-day period. Twelve Aspergillus isolates including 11 A. parasiticus and 1 A. flavus produced aflatoxins which were extracted from both the mycelium and culture filtrate. Aflatoxin production was detected at day 3 and was detected continually for up to day 20. Aflatoxin B1 production was greatest between 7 and 10 days and significantly higher quantities were produced by A. flavus compared to A. parasiticus. Aflatoxin production was not a stable trait in 1 A. parasiticus isolate passaged 50 times on agar. In addition to loss of aflatoxin production, an associated loss in sporulation ability was also observed in this passaged isolate, although it did maintain pathogenicity against S. sacchari. An aflatoxin B1 concentration of 0.16 micrograms/mealybug (14.2 micrograms/g wet wt) was detected within the tissues of infected mealybugs 7 days after inoculation. In conclusion, the ability of Aspergillus isolates to produce aflatoxins was not essential to the entomopathogenic activity of this fungus against its host S. sacchari.     

Biodiversity of the bacterial flora on the surface of a smear cheese

The bacteria on the surface of a farmhouse smear-ripened cheese at four stages of ripening (4, 16, 23, and 37 days) from inoculated (i.e., deliberately inoculated with Brevibacterium linens BL2) and noninoculated (not deliberately inoculated with B. linens BL2) cheese were investigated. The results show that, contrary to accepted belief, B. linens is not a significant member of the surface flora of smear cheese and no microbial succession of species occurred during the ripening of the cheeses. Of 400 isolates made, 390 were lactate-utilizing coryneforms and 10 were coagulase-negative Staphylococcus spp. A detailed analysis of the coryneforms was undertaken using phenotypic analysis, molecular fingerprinting, chemotaxonomic techniques, and 16S rRNA gene sequencing. DNA banding profiles (ramdom amplified polymorphic DNA [RAPD]-PCR) of all the coryneform isolates showed large numbers of clusters. However, pulsed-field gel electrophoresis (PFGE) of the isolates from the cheeses showed that all isolates within a cluster and in many contiguous clusters were the same. The inoculated and noninoculated cheeses were dominated by single clones of novel species of Corynebacterium casei (50.2% of isolates), Corynebacterium mooreparkense (26% of isolates), and Microbacterium gubbeenense (12.8% of isolates). In addition, five of the isolates from the inoculated cheese were Corynebacterium flavescens. Thirty-seven strains were not identified but many had similar PFGE patterns, indicating that they were the same species. C. mooreparkense and C. casei grew at pH values below 4.9 in the presence of 8% NaCl, while M. gubbeenense did not grow below pH 5.8 in the presence of 5 to 10% NaCl. B. linens BL2 was not recovered from the inoculated cheese because it was inhibited by all the Staphylococcus isolates and many of the coryneforms. It was concluded that within a particular batch of cheese there was significant bacterial diversity in the microflora on the surface.     

Inhibition of Listeria monocytogenes by enterocin 4 during the manufacture andripening of Manchego cheese

The inhibitory effect of enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4, on Listeria monocytogenes strains Ohio and Scott A during themanufacture and ripening of Manchego cheese was investigated. Raw ewe's milk wasinoculated with ca 10⁵ cfu ml⁻¹ of L.monocytogenes and with 1% of a commercial lactic starter, 1% of an Ent. faecalis INIA 4 culture, or 1% of each culture. Manchego cheeses were manufactured according tousual procedures. Listeria monocytogenes Ohio counts decreased by 3 log units after8 h and by 6 log units after 7 d in cheese made from milk inoculated with Ent. faecalis INIA 4 or with both cultures, whereas no inhibition was recorded after 60 d in cheese made frommilk inoculated with commercial lactic starter. Listeria monocytogenes Scott A wasnot inhibited by enterocin 4 during cheese manufacture, but decreases of 1 log unit after 7 d andof 2 log units after 60 d were achieved in cheese made from milk inoculated with bothcommercial lactic starter and Ent. faecalis INIA 4.     

Surface microflora of four smear-ripened cheeses

The microbial composition of smear-ripened cheeses is not very clear. A total of 194 bacterial isolates and 187 yeast isolates from the surfaces of four Irish farmhouse smear-ripened cheeses were identified at the midpoint of ripening using pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR, and 16S rRNA gene sequencing for identifying and typing the bacteria and Fourier transform infrared spectroscopy and mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) analysis for identifying and typing the yeast. The yeast microflora was very uniform, and Debaryomyces hansenii was the dominant species in the four cheeses. Yarrowia lipolytica was also isolated in low numbers from one cheese. The bacteria were highly diverse, and 14 different species, Corynebacterium casei, Corynebacterium variabile, Arthrobacter arilaitensis, Arthrobacter sp., Microbacterium gubbeenense, Agrococcus sp. nov., Brevibacterium linens, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus saprophyticus, Micrococcus luteus, Halomonas venusta, Vibrio sp., and Bacillus sp., were identified on the four cheeses. Each cheese had a more or less unique microflora with four to nine species on its surface. However, two bacteria, C. casei and A. arilaitensis, were found on each cheese. Diversity at the strain level was also observed, based on the different PFGE patterns and mtDNA RFLP profiles of the dominant bacterial and yeast species. None of the ripening cultures deliberately inoculated onto the surface were reisolated from the cheeses. This study confirms the importance of the adventitious, resident microflora in the ripening of smear cheeses.     

Aflatoxin is degraded at different temperatures and pH values by mycella of Aspergillus parasiticus

Summary Blended 9-day-old mycelia of Aspergillus parasiticus NRRL 2999 were tested for their ability to degrade aflatoxins B₁ and G₁ at 7,19,28,36, and 45°C. Rates for degradation of aflatoxin B₁ and G₁ were maximum at 28°C. Intermediate rates of aflatoxin degradation were observed at 19 and 36°C while little aflatoxin was degraded at 7 and 45°C. Five different pH values (2.0, 3.0, 4.0, 5.0, and 6.5) were also tested to determine the effect of pH on ability of blended 9-day-old mycelia of A. parasiticus NRRL 2999 to degrade aflatoxins. The ability of mycelia to degrade aflatoxin was pH-dependent. Of the pH values tested, greatest rates of aflatoxin B₁ and G₁ degradation occurred when pH was in the range of 5 to 6.5. Little aflatoxin was degraded at pH 4.0 and essentially no aflatoxin was degraded by mycelia at pH 2.0 or 3.0 although some aflatoxin was degraded by acid conditions only at pH values of 4 or less.