Effect of Temperature Cycling on the Production of Patulin and Citrinin

Two strains of Peniciltium urticae (NRRL 2159A and 1952) and one strain of P. cltrinun (NRRL 5927) were used to investigate the effect of temperature Oycling on the production of patulin and citrinin. Temperature cycling (33–25°C, 33–15°C, and 25–15°C) delayed the rate of patulin biogenesis. As a general rule, cultures grown under cycling temmperature conditions produced less patulin than cultures grown a1 constant temperatures. The effect of temperature cycling (30–25°C, 30–15°C, and 25–15°C) on citrinin production was found to vary with the temperature combination.     

Public Health Significance of Molds and Mycotoxins in Fermented Dairy Products

Mold growth on cheese and other fermented dairy products is a common and recurring problem. Potential mycotoxin contamination is serious since some molds can grow and produce mycotoxins at temperatures as low as ?2 to 10°C. Work can be divided into: 1) incidence, types, and mycotoxin-producing potential of molds in fermented dairy products, 2) experimental mycotoxin production on cheese under conditions of storage and aging of cheese, 3) natural occurrence of mycotoxins in commercial samples of cheese, and 4) potential toxicity of Penicillium roqueforti and its significance in blue veined cheeses.Molds most common on cheese and fermented dairy products are Penicillium species. Mycotoxins produced by these organisms are penicillic acid, patulin, ochratoxin A, and citrinin. Percentages of molds in cheese capable of producing some commonly studied mycotoxins ranged from 1.8% to 12.4%. Cheese is an excellent substrate for mold growth but a poor substrate for mycotoxin production. Several natural occurrences of mycotoxins in cheese include small and variable amounts of patulin, penicillic acid, sterigmatocystin (600 µg/kg), penitrem A, and mycophenolic acid. Penicillium roqueforti is capable of producing toxic alkaloids and other compounds. The significance of these substances for human health is unclear.The decision to trim or to discard moldy cheese can be aided by considering the risk versus benefit based on storage history (temperature), extent of mold growth, appearance of mold (color), and size of cheese.     

Survival of Penicillium expansum and Patulin Production on Stored Apples after Wash Treatments

ABSTRACT: Penicillium expansum is a widespread fungus found on apples that causes fruit decay and may lead to production of a toxic secondary metabolite, patulin. This study was undertaken to evaluate the effectiveness of several chemical sanitizers against P. expansum NRRL 2304 and to establish sanitizing wash treatments that would inhibit P. expansum growth and subsequent patulin production on Empire apples destined for cider. Wash treatments included 200 ppm NaOCl, 1% StorOx®, 0.5% potassium sorbate, 300 ppm SO₂, and 0% to 5% acetic acid. Spores of P. expansum or inoculated apple slices were dipped in sanitizing wash solution for 5 min, and mold growth and patulin production was monitored on subsequent storage. It was found that 0.5% potassium sorbate and 300 ppm SO₂ did not affect mold survival or patulin production; 1% StorOx® was effective against mold spores in solution (4 log Most Probable Number destruction of spores), but there was no significant reduction in spore count when the same solution was used to sanitize mold‐inoculated apple discs. Washing with 200 ppm NaOCl delayed growth of P. expansum on inoculated apple discs but failed to completely inhibit patulin production. Acetic acid solution (2% to 5%) was the most efficient chemical against P. expansum. A wash treatment with ≥2% acetic acid for more than 1 min is recommended to completely inhibit growth of P. expansum and subsequent patulin production on apples destined for cider.     

Mycotoxins in foodstuffs. XII. The influence of the water activity (a-w) of cakes on the growth of moulds and the formation of mycotoxins.

The influence of various a-w values (high, moderate and low, e.g. 0.92, 0.87, and 0.82) of cakes ("Rührkuchen", made of wheat flour, sugar, eggs, fat and water) on the development and the mycotoxin production of Aspergillus flavus (aflatoxins), A. versicolor (sterigmatocystin), A. ochraceus (ochratoxin A), Penicillium chrysogenum (citrinin) and P. expansum (patulin) was investigated. None of the moulds was able to germinate at an a-w of 0.82. An increase of the water activity resulted in a pronounced growth of the five fungi. Citrinin was not formed on the cakes at all. The yields of the other mycotoxins were not markedly influenced by the a-w (0.92 or 0.87). During the development of the moulds the a-w of the substrate was lowered at first (water requirement during germination and initial growth) and subsequently increased (initiation of the secondary metabolism).     

Production of mycotoxins by Penicillium expansum inoculated into apples

We investigated the production of mycotoxins in apple fruits inoculated with spores of 40 strains of apple blue mold, Penicillium expansum. Patulin and citrinin contents in the extracts from apples stored at 25 degrees C for 12 days after inoculation were determined by high-performance liquid chromatography (HPLC) analysis with UV and fluorescence detection. Patulin and citrinin were produced by 90% (36) and 80% (32) of the 40 strains, indicating that P. expansum is a consistent producer of these mycotoxins. The patulin content in the extracts was substantially higher than the citrinin content. Other mycotoxins whose production in pure culture has been reported were simultaneously detected with high-resolution liquid chromatography-mass spectrometry (LC-MS) analysis with the positive ion mode of electrospray ionization. Along with patulin and citrinin, expansolides A and B were identified based on the HPLC and LC-MS spectral data and detected in 88% (35) of the extracts. The results indicate that P. expansum is a consistent producer of expansolides A and B in rotten areas of apple fruits. The findings raise the possibility that products from decayed apples might contain expansolides A and B in addition to patulin and citrinin.     

Production and stability of patulin, ochratoxin A, citrinin, and cyclopiazonic acid on dry cured ham

Toxinogenic fungal species can be isolated from dry cured meat products, raising the problem of the direct contamination of these foods by mycotoxins known to be carcinogenic or potent carcinogens. Because the contamination of a food by mycotoxins can be considered a balance between production and degradation, the stability of mycotoxins on dry cured meat was also investigated. This study focused on patulin, ochratoxin A, citrinin, and cyclopiazonic acid that can be produced by fungal species previously isolated from dry cured meat products sold on the French market. We demonstrated that neither patulin nor ochratoxin A was produced on dry meat by toxigenic strains, whereas relatively high amounts of citrinin and cyclopiazonic acid were found after a 16-day incubation period at 20 degrees C (87 and 50 mg/kg, respectively). After direct contamination, the initial content of patulin rapidly decreased to become undetectable after only 6 h of incubation at 20 degrees C. For both citrinin and ochratoxin A, the kinetics of decrease at 20 degrees C was less rapid, and the two toxins presented half-lives of 6 and 120 h, respectively. By contrast, more than 80% of the initial contamination in cyclopiazonic acid was still found on ham after a 192-h incubation period. Toxin stability was not affected by storage at 4 degrees C. These results suggest that growth of toxigenic strains of Penicillium has to be avoided on dry meat products.     

Saccharomyces cerevisiae YE‐7 reduces the risk of apple blue mold disease by inhibiting the fungal incidence and patulin biosynthesis

The yeast strain, Saccharomyces cerevisiae YE‐7, was tested to control the apple blue mold disease caused by Penicillium expansum and the accumulation of patulin mycotoxin. Compared with control, pre‐treatment of pathogen‐infected apples with YE‐7 significantly reduced the incidence of blue mold diseases and patulin accumulation in rotten apple tissue by 48% and 42.6%, respectively. Interestingly, late treatment of pathogen‐infected apples with YE‐7 did not decrease patulin accumulation in the rotten tissue compared with pre‐treatment or simultaneous‐treatment. Independently, patulin amount and gene expression of isoepoxydon dehydrogenase were also measured. YE‐7 pre‐treated apples showed 75% and four times reduction in patulin accumulation and IDH gene expression, respectively. The results indicated that YE‐7 directly affected patulin biosynthesis and did not affect accumulated patulin. On the other hand, cold storage enhanced the effect of YE‐7 on apple blue mold disease incidence.

Practical applications

Postharvest blue mold decay caused by Penicillium expansum was important postharvest disease of apples. In addition, under favorable conditions, P. expansum can cause severe losses in apple fruits due to the production of patulin. In this study, a strain of S. cerevisiae (YE‐7) was assessed for its efficacy not only in controlling apple blue mold diseases, but also in reducing patulin accumulation in fruit tissue. The research will give a practical way to control the decay in a bio‐control way instead of the chemical.     

Some factors influencing patulin production by Penicillium expansum in pome fruits

BACKGROUND: The objective of our study was to examine the effects of Penicillium expansum on patulin production in relation to isolates, species and cultivar type, incidence and severity of decay. In addition, patulin production at different incubation times and its diffusion were also investigated. These factors were evaluated in pome fruits inoculated with P. expansum and kept at 20 °C for short periods of time. RESULTS: The ability of five P. expansum isolates to grow and produce patulin in inoculated Golden Delicious apples varied among the strains from below the limit of quantification to 662 µg kg^?1. Variety and species of pome fruits influenced patulin production. P. expansum isolate PE97.IT produced a higher patulin content in apples than in pears. The highest patulin production was 386 µg kg^?1 in Golden Delicious. No blue mould symptom appeared in pears inoculated with P. expansum and no patulin was detected after 3 days at 20 °C. However, patulin increased with incubation time after 6 and 8 days. No patulin was detected in healthy pear tissue but it was high in the decayed area. CONCLUSION: Since patulin production is associated primarily with infected rotten tissue, patulin control is possible by using healthy fruits, sorting damaged and rotten fruits before processing. Copyright © 2010 Society of Chemical Industry     

Secondary metabolism in Penicillium expansum: Emphasis on recent advances in patulin research

The plant pathogenic fungus Penicillium expansum is a major concern of the global food industry due to its wide occurrence and ability to produce various mycotoxins, of which the most significant is patulin. Relatively less highlighted in the literature, in comparison with the other food-borne mycotoxins, patulin is one of the main factors in economic losses of vegetables and fruits. Otherwise, patulin is a health hazard which results in both short-term and long-term risks. This review includes knowledge on the biosynthetic mechanisms used for secondary metabolite production in P. expansum, with special emphasis on patulin biosynthesis. The abiotic factors triggering the production of patulin and the strategies developed to reduce or prevent the contamination by this mycotoxin are comprehensively discussed. The database presented in this review would be useful for the prioritization and development of future research.     

CONTAMINATION OF GRAINS BY MYCOTOXIN-PRODUCING MOLDS AND MYCOTOXINS AND CONTROL BY GAMMA IRRADIATION

Ninety random grain samples were collected and analyzed for mycotoxins, and the effect of gamma irradiation on the production of mycotoxins in grains was studied. Aspergillus, Penicillium, Mucor, Rhizopus, Fusarium, Alternaria, Scopulariopsis and Cladosporium were the most common fungal genera isolated from grains. Aspergillus flavus, Aspergillus niger, Aspergillus candidus, Aspergillus ochraceus, Penicillium citrinum, Penicillium expansum, Penicillium citreonigrum, Penicillium purpurogenum, Penicillium griseofulvum and Penicillium verrucosumwere the most common Aspergillus and Penicillium species in grains. Out of 120 Aspergillus and Penicillium isolates, 80 were mycotoxin producers. Analysis of grains revealed the occurrence of aflatoxin B₁ ochratoxin A, cycolopiazonic acid and citrinin. Of the 90 samples, 67 were positive for one or more mycotoxin. Irradiation of grains at dose of 2.0 and 4.0 kGy decreased significantly the total fungal counts compared with unirradiated controls. After 100 days of storage at room temperature, the unirradiated grains were contaminated with high concentrations of mycotoxins as compared with irradiated 4.0‐kGy samples. Mycotoxin production in grains decreased with increasing irradiation doses and was not detected at 6.0 kGy over 100 days of storage.     

Ergosterol and mycotoxins in grain dusts from fourteen Belgian cereal storages: A preliminary screening survey

Grain dusts from farms and storage companies are generally used in animal feeding. They can give rise to airborne dust in the environment of workers or accidentally contaminate the following stored grains. Because dusts are potential mycotoxin‐rich materials, this preliminary screening survey was undertaken on citreoviridin, citrinin, cyclopiazonic acid, deoxynivalenol, gliotoxin, helvolic acid, mycophenolic acid, nivalenol, ochratoxin A, patulin, penicillic acid, secalonic acid D, sterigmatocystin, zearalenol and zearalenone. Furthermore, ergosterol was determined as a fungal growth marker. Fourteen grain dusts collected from farms and storage companies in Belgium were assayed and toxins co‐occurred at uneven distributions with wide ranges of concentrations. Median concentrations exceeded 1 µg g^?1 for penicillic acid, gliotoxin, deoxynivalenol, helvolic acid, mycophenolic acid, patulin, sterigmatocystin and zearalenol. Using the median values, an assessment of worker exposure indicated that mycotoxin uptake through dust inhalation may simultaneously contribute to 0.5, 0.5, 0.7 and 0.1% of the respective tolerable daily intake of ochratoxin A, patulin, deoxynivalenol and zearalenone. In spite of these low values, the question of multi‐contamination of grain dusts and, consequently, exposure to several mycotoxins should not be underestimated. Moreover, inhalation of contaminated airborne aerosols can represent an additional route of exposure which has not been exhaustively investigated so far. Copyright © 2007 Society of Chemical Industry     

红曲霉合成莫纳克林K的研究进展

红曲霉是一种药食两用丝状真菌,广泛应用于食品、医药、生物催化等领域。莫纳克林K是红曲霉主要代谢功能活性产物,具有降脂、抗疲劳、抗菌、抗癌和保护神经等生物活性,是公认的降低胆固醇的理想药物。研究对比了红曲霉与土曲霉合成莫纳克林K基因簇的不同,阐述了红曲霉产莫纳克林K的合成途径;对莫纳克林K在医药和保健食品、饲料等领域中应用的情况和潜力进行了归纳和展望。由于天然红曲霉发酵产功能性物质过程中,伴随产生的真菌毒素桔霉素,对肾脏、肝脏、心脏和生殖系统有毒性影响,使红曲霉工业化发展受到限制。提高红曲霉的莫纳克林K产量,并降低桔霉素的产生,是解决功能红曲发展问题的重要途径。因此,从诱变育种、构建工程菌株、优化培养基、控制环境因子等方面总结了优选红曲霉高产莫纳克林K并避免桔霉素积累的技术方法。主要对红曲霉产莫纳克林K的合成途径、生物活性及应用领域、高产莫纳克林K的生物技术方法研究现状进行总结,在此基础上分析红曲霉产莫纳克林K中存在的问题,并提出建议与展望,以期为新型红曲保健食品的研究与开发提供理论依据和参考。     

Potential mycotoxin problems in mould-fermented sausage

Summary A considerable proportion of the sausage consumed in many European countries is produced by a mould-fermentation process that does not involve pure culture technics.422Penicillium cultures isolated from mould-ripened sausages produced in II European countries were analyzed for their ability to produce the following nine mycotoxins: aflatoxin B₁ and G₁ ochratoxin A, penicillic acid, patulin, citrinin, tremortin A, zearalenone, rubratoxin B; 88 isolates were found capable of toxin synthesis (20.9%). 44 of these cultures produced penicillic acid, 17 ochratoxin A, 11 tremortin A, 10 citrinin, and 6 patulin. 3 cultures produced both patulin and citrinin.Sausages were ripened with 6 moulds producing penicillic acid, 17 producing ochratoxin, 5 producing citrinin and 3 each producing patulin or tremortin. No mycotoxins were detected up to 70 days of ripening.Direct addition of penicillic acid to raw sausage resulted in disappearance of the mycotoxin. Amino acids normally occuring in meat were found capable of rapidly reacting with penicillic acid to produce adducts that were nontoxic to laboratory animals.Although our data indicate that consumption of mould ripened sausage is not a health hazard with respect to the 9 mycotoxins analyzed for, it is recommended that manufacturers of these products adopt pure culture technics using moulds known to be toxicologically safe.

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Zusammenfassung In zahlreichen Ländern Europas werden seit altersher mit Schimmelpilzen gereifte Rohwürste hergestellt. Es handelt sich hierbei um traditionelle Produktionsverfahren, bei denen jedoch keine Schimmelpilz-Reinkulturen verwendet werden.Wir untersuchten das Toxinbildungsvermögen von 422Penicillium-Stämmen, die von schimmelpilzgereiften Rohwürsten isoliert worden waren, die aus II verschiedenen Ländern stammten. Insbesondere interessierten uns die Mykotoxine Aflatoxin B₁ und G₁, Ochratoxin A, Penicillinsäure, Patulin, Citrinin, Tremortin A, Zearalenon und Rubratoxin B. Bei 88 Stämmen (20,9%) war eine Toxinbildung nachweisbar. 44 dieser Kulturen bildeten Penicillinsäure, 17 Ochratoxin A, 11 Tremortin A, 10 Citrinin und 6 Patulin. Von drei Stämmen wurde sowohl Patulin als auch Citrinin gebildet.Wurden frisch hergestellte Rohwürste mit Schimmelpilzen, die die genannten Toxine bilden, beimpft, so konnten während der 70 Tage dauernden Reifung keine Mykotoxine in der Wurst nachgewiesen werden.Den Rohwürsten direkt zugesetzte Penicillinsäure wurde inaktiviert, da dieses Mykotoxin sehr rasch mit Aminosäuren reagiert, die im Fleisch vorhanden sind.Obwohldiese Untersuchungen darauf hinweisen, daß sehimmelpilzgereifte Rohwürste, zumindest bezüglich der 9 genannten Mykotoxine, keine potentielle Gefahr für den Verbraucher darstellen, sollte man dennoch dazu übergehen als Starterkultur nur technologisch und toxikologisch geprüfte Schimmelpilz-Stämme in Reinkultur einzusetzen.

     

Some factors governing the production of patulin in apples

The production of patulin by Penicillium patulum and P. expansum growing on apple juice and apple tissue derived from Bramley's seedling apples has been investigated. The carbon sources responsible for the patulin production have been determined using ¹⁴C techniques and growth on defined media.     

Mycotoxin production by molds isolated from ‘Greek-style’ black olives

Seventeen mold strains were isolated from ‘Greek-style’ black olives produced in Morocco. Eight of these isolates were identified as Aspergillus flavus, seven as Aspergillus petrakii, and two as Aspergillus ocharaceus Wilhelm. The A. flavus strains were tested for production of aflatoxins B₁, B₂, G₁, and G₂; and A. ochraceus and A. petrakii strains were tested for production of ochratoxin, penicillic acid, patulin, and citrinin. The organisms were tested for mycotoxin production on five different substrates, including rice powder-corn steep agar, autoclaved rice, yeast-extract sucrose broth (YES), potato dextrose agar (PDA), and fresh olive paste. All strains of A. flavus produced aflatoxins on all substrates except olive paste and PDA. In PDA, only two strains produced Aflatoxin B₁. Five A. ochraceus group isolates produced penicillic acid on one or more of the substrates, but only two out of the five produced penicillic acid on olive paste. None produced ochratoxin, patulin or citrinin. Quantities of aflatoxin B₁ produced in rice ranged from 5 to 14 μg/g of rice, and of penicillic acid 15–32 μg/g of rice. In olive paste, the concentrations of penicillic acid were 11.4 and 30.2 μg/g. Biological toxicity of extracts of mold cultures was confirmed using chicken embryos and a microbiological test. Crude extracts of cultures were also tested for mutagenicity using the Salmonella mutagenicity (Ames) Test, and some gave positive mutagenic responses.     

The production of ochratoxin A and citrinin in barley

The production of ochratoxin A by Aspergillus ochraceus and of ochratoxin A and citrinin by Penicillium viridicatum growing on previously sterilised barley for 200 days at 5, 10 and 20°C and a water activity of 0.85 is reported. A. ochraceus did not grow at 5°C, multiplied slowly at 10°C but did not produce toxin. At 20°C the organism multiplied more quickly and produced ochratoxin after 19 days, which slowly disappeared over the next 150 days. P. viridicatum grew slowly at 5°C but did not produce any toxin. It multiplied at 10°C and produced ochratoxin A which was only detectable during the period from 100 to 150 days. At 20°C both ochratoxin A and citrinin were produced. Ochratoxin A was detected after 10 days and was still present after 240 days, whereas citrinin was produced in large quantities between 118 and 129 days and then rapidly disappeared.     

Evaluation of several culture media for production of patulin by Penicillium species

The aim of this study was to evaluate different species of Penicillium to identify those which have the potential to produce the greatest amount of the mycotoxin, patulin. Additionally, six different culture media were compared to determine maximum patulin production. Eleven different strains of Penicillium species were selected because they had previously been reported to be producers of patulin. The strains included Penicillium expansum, Penicillium griseofulvum (formerly Penicillium urticae), Penicillium clavigerum, and Penicillium coprobium and a recent Penicillium sp. isolated from an apple. Cultures were grown in duplicate in three different liquid media: potato dextrose, malt extract, and glucose/yeast extract/peptone, both with and without manganese supplementation. Patulin production was compared at 24, 48, 72, and 96 h. Variability in patulin production occurred among the different species, growth media used, and time of incubation. All three of the P. griseofulvum isolates were the highest producers of patulin at 96 h. For most of the strains, potato dextrose broth supplemented with manganese was optimal for maximum production of patulin. Although P. expansum is frequently cited as the most likely source of patulin in apple juice, certain other Penicillium species are capable of producing more patulin than strains of P. expansum. The apple juice industry should be alert to the possibility that Penicillium species other than P. expansum can be responsible for the occurrence of patulin.     

Biomass and Patulin Production by Byssochlamys nivea in Apple Juice as Affected by Sorbate, Benzoate, SO2 and Temperature

The effects of potassium sorbate, sodium benzoate, SO₂ and incubation temperature on biomass and patulin production by Byssochlamys nivea in apple juice were determined. Growth at 21, 30 and 37°C over a 25-day incubation period was significantly retarded by 75 ppm SO₂, 150 ppm potassium sorbate and 500 ppm sodium benzoate. Biomass accumulated to approximately 500 mg/100 ml in control samples of apple juice. Patulin was produced in the highest concentrations at 21°C after 20 days incubation. After reaching a maximum concentration at 30 and 37°C, a rapid decline in patulin content was observed. Patulin production was also observed at 12°C. On the basis of concentration, SO₂ had the most significant effect on the rate of biomass and patulin production by B. nivea followed by potassium sorbate and sodium benzoate, respectively.     

Carcinogenic and genotoxic effects of mycotoxins

Aflatoxin B1 is the most potent hepatocarcinogen among the naturally occurring compounds. It's carcinogenic effect was confirmed by longterm-studies in several animal species. There exists detailed knowledge on the molecular mechanism of the carcinogenic effect caused by aflatoxin B1. At present there does not exist a reliable evidence that human primary hepatocellular carcinoma is caused by aflatoxins. Sterigmatocystin is a hepatocarcinogen too. The genotoxicity of sterigmatocystin likewise as of aflatoxin B1 was detected by short-term-tests with microbial systems and mammalian cells in vitro. It is disputable whether ochratoxin A, citrinin and T-2 toxin are carcinogenic or not. Diacetoxyscirpenol, zearalenone and patulin are estimated as not carcinogenic after longterm-studies with oral toxin administration. Patulin and penicillic acid are genotoxic in microbial systems respectively in mammalian cells in vitro. Luteoskyrin is carcinogenic, but there was not detected any genotoxicity.