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.     

Potential aflatoxin hazards to human health from direct mold growth on Teleme cheese

Ninety-four commercial Teleme cheese samples were examined for aflatoxins produced by direct mold growth. The mycoflora on the cheese and in the atmosphere of Teleme cheese plants also were monitored. Penicillium and Aspergillus genera were tested for aflatoxin production after growth on Teleme cheese at 25 degrees C. In all cases, over 78% of the molds were Penicillium species. Aspergillus made up 3.8 to 3.9% and 0 to 7.3% of the mold on cheese and in the plant atmosphere, respectively. None of the commercial samples contained aflatoxins and none of the 448 Penicillium isolates was an aflatoxin producer. Of 22 Aspergillus species, one was capable of producing aflatoxins after direct growth on cheese. Because the physicochemical characteristics of Teleme cheese (high moisture, low pH, and medium salt concentration) favor mold growth, care should be taken to avoid contamination of the cheese by aspergilli.     

MOLD GROWTH AND PRESENCE OF AFLATOXIN IN SOME TURKISH CHEESES

Twenty-five random fresh market samples of Van herby cheese and pickled white cheese were examined for molds and aflatoxins. The mean total mold count in Van herby cheese was 2.50 × 10⁵/g; in pickled white cheese it was 4.95 × 10⁴/g. The mycoflora on the cheeses were determined. In all cheeses, over 65% of molds were Penicillium species . Aspergillus made up 0 to 1.6 % and 2.6 % to 4.0 % of the mold on pickled white cheese and Van herby cheese, respectively. Other isolated molds belonged to Mucor, Geotrichum and Trichoderma genera. None of the samples contained aflatoxins and none of the 6 Aspergillus isolates was an aflatoxin producer .     

OCCURRENCE OF MOLDS IN FRESH CHEESES

The purpose of our work was isolation, enumeration and identification of molds in fresh cheeses. A total of 112 cheese samples were obtained from different retail markets in Santa Cruz de Tenerife. Of the samples, 57.2% were positive for industrially craft produced cheeses. The values for the industrially produced cheeses ranged between 10² and 10⁶ CFU/g, for the craft produced cheeses the corresponding values were 10²-10⁷ CFU/G and 4.4x103. The genera identified most often in both types of cheeses were Geotrichum and Penicillium with 49.4 and 29.1% of isolates, respectively. Species of the Aspergillus genus, the main producer of aflatoxins, was detected in 1.3% of the total samples analyzed. Nonetheless, the presence of such genera as Fusarium, Cephalosporium, Cladosporium, etc. is considered to be undesirable, as they are capable of producing other mycotoxins related to specific pathological processes.     

Roquefortine C occurrence in blue cheese

Several strains of Penicillium are used for the production of mold-ripened cheeses, and some of them are able to produce mycotoxins. The aims of the research were the determination of roquefortine C and PR toxin in domestic and imported blue cheeses, the identification of the penicillia used as starter, and the investigation of their capacity for producing toxins in culture media. Roquefortine C was always found in the cheeses at levels ranging from 0.05 to 1.47 mg/kg, whereas the PR toxin was never found. The identification of the fungal strains present in the domestic cheeses included Penicillium glabrum, Penicillium roqueforti, and Penicillium cyclopium in the Gorgonzola "dolce" and Penicillium roqueforti in the Gorgonzola "naturale"; in one case, the presence of Penicillium crustosum was observed. The strains isolated from the foreign cheeses belonged to P. roqueforti. The strains were able to produce between 0.18 and 8.44 mg/liter of roquefortine in yeast extract sucrose medium and between 0.06 and 3.08 mg/liter and less than 0.05 mg/liter when inoculated in milk at 20 degrees C for 14 days and 4 degrees C for 24 days, respectively. Linear relations between production of roquefortine in culture media and cheeses did not emerge. PR toxin ranged from less than 0.05 to 60.30 mg/liter in yeast extract sucrose medium and was produced in milk at 20 degrees C from only one strain. The low levels and the relatively low toxicity of roquefortine make the consumption of blue cheese safe for the consumer.     

Filamentous Fungi and Mycotoxins in Cheese: A Review

Important fungi growing on cheese include Penicillium, Aspergillus, Cladosporium, Geotrichum, Mucor, and Trichoderma. For some cheeses, such as Camembert, Roquefort, molds are intentionally added. However, some contaminating or technological fungal species have the potential to produce undesirable metabolites such as mycotoxins. The most hazardous mycotoxins found in cheese, ochratoxin A and aflatoxin M1, are produced by unwanted fungal species either via direct cheese contamination or indirect milk contamination (animal feed contamination), respectively. To date, no human food poisoning cases have been associated with contaminated cheese consumption. However, although some studies state that cheese is an unfavorable matrix for mycotoxin production; these metabolites are actually detected in cheeses at various concentrations. In this context, questions can be raised concerning mycotoxin production in cheese, the biotic and abiotic factors influencing their production, mycotoxin relative toxicity as well as the methods used for detection and quantification. This review emphasizes future challenges that need to be addressed by the scientific community, fungal culture manufacturers, and artisanal and industrial cheese producers.     

FUNGAL BIOTA ISOLATED FROM SPANISH CHEESES

Fungal biota, with special reference to the genus Penicillium, was studied in 52 samples of commercial cheeses (10 fresh, 17 semiripened and 25 ripened) made from different types of milk (cow, ewe, goat and mixed) produced in southern Spain. In 41 of the total of cheeses analyzed (79%) molds were isolated. Penicillium was identified in 63% of the samples , Mucor spp. in 27% , Geotrichum candidum in 17% and Cladosporium herbarum in 10%; eleven other fungal genera were detected ranging from 2 to 4%. Thirty-five species of Penicillium were analyzed with the following distribution: 7 in fresh cheese, 16 in semiripened cheese and 30 in ripened cheese. The incidence of Penicillium spp. was also greater in the cheeses with a higher degree of ripeness, i.e. 20% in fresh cheese, 71% in the semiripened and 76% in the ripened cheese.     

Nonspecific Enzyme-Linked lmmunosorbent Assay for Molds in Foods

A nonspecific enzyme-linked immunosorbent assay (ELISA) was developed to detect molds in foods by producing an antibody to a mixture of six common molds, Aspergillus versicolor, Cladosporium herbarum, Fusarium poae, Geotrichum candidum, Mucor circinelloides, and Penicillium chrysogenum. This antibody recognized these mold genera plus 10 others but not yeasts. Mold antigens added into Cheddar and cottage cheeses, fruit juices, nonfat dry milk, raisins, and yogurt could be detected. Molds (10² spores/g) inoculated into cottage cheese and yogurt and allowed to grow at 7 or 22°C were detected at 10³ CFU/g. A nonspecific ELISA could be developed to detect general mold contamination of foods.     

进口干酪中霉菌污染的调查

依据GB4 789.15- 94方法 ,对 133件进口干酪进行了霉菌总数的测定和菌相分析。86 .4 7%的样品无霉菌污染 ,但从 18件样品中检出了霉菌 (霉菌总数 >10CFU/ g) ,检出率为13.53%。不同种类干酪的检出率无差别。从 133件干酪中分离出 10 6株霉菌 ,青霉占被分离菌株的 6 8.87% ,其中大部分为娄地青霉。硬质干酪中优势菌群为娄地青霉和常现青霉 ,软质及半软质干酪中优势菌群为拟青霉和娄地青霉。由此可见 ,加强进口干酪的霉菌检测是十分必要的。     

Citrinin production and stability in cheese

Citrinin is a nephrotoxic fungal metabolite that has been demonstrated to be mutagenic in hepatocytes. It can be produced by several fungal species that belong mainly to the genus Penicillium and has been isolated from many feeds and human foods. Cheese is a very sensitive product because it can be naturally contaminated by citrinin-producing molds. The purpose of this study was to determine whether citrinin can be produced in cheeses and whether it is stable in these products. Both toxigenic strains of Penicillium citrinum and Penicillium expansum used were able to produce citrinin in cheese at 20 degrees C, but not at 4 degrees C. Up to 600 mg of citrinin per kg of cheese was obtained after 10 days of incubation. Interestingly, fresh goat cheese appeared to be a more favorable substrate for toxigenesis than did yeast extract-sucrose medium. Although contamination was mainly superficial, 33% of the toxin remained in cheese after trimming. Moreover, citrinin appeared to be very stable in some of the tested cheeses (goat cheese, Saint Marcellin, Soignon). For all cheeses tested, more than 50% of the initial content of citrinin was still present after 8 days of storage. Taken together, these results suggest that the contamination of cheeses by wild strains of Penicillium must be avoided.     

Survey of the mycobiota of Spanish malting barley and evaluation of the mycotoxin producing potential of species of Alternaria, Aspergillus and Fusarium

The present work deals with the toxigenic mycobiota occurring in Spanish malting barley and the capability for producing mycotoxins by several important toxigenic fungi. One hundred and eighty seven samples of malting barley were gathered from Spanish breweries before processing. One hundred and fifty kernels per sample were surface-sanitized with a 2% sodium hypochlorite solution and incubated on three culture media. The most abundant fungi were species of Alternaria, Aspergillus, Penicillium and Fusarium, which were present in 93%, 82.3%, 57.8% and 27.8% of the samples, respectively. To evaluate their mycotoxin producing potential a number of isolates belonging to each genus, except Penicillium, were randomly selected and incubated on culture media known to be appropriate for production of mycotoxins. Alternariol and alternariol monomethyl ether were produced by 26.7% of Alternaria spp. isolates (all belonged to Alternaria alternata). All tested isolates of F. verticillioides produced fumonisin B(1) (FB(1)) and 61.3% of them produced fumonisin B(2) (FB(2)), whereas FB(1) was synthesized by 83.3% and FB(2) by 77.8% of F. proliferatum isolates. Twenty percent of the isolates of the Aspergillus flavus/A. parasiticus group had the capability to produce aflatoxin B(1) and aflatoxin B(2). Thirty out of 34 isolates of F. graminearum produced deoxynivalenol and zearalenone whereas the other 4 isolates produced nivalenol. Ochratoxin A was detected in 75% and 15% of isolates of Aspergillus section Nigri and A. ochraceus, respectively. This is the first survey carried out in Spain on the toxigenic mycobiota contaminating malting barley in breweries and the mycotoxin producing capacity of several species. The information obtained is useful for assessing the risk of mycotoxins in beer.     

Toxigenic penicillia spoiling frozen chicken nuggets

Frozen chicken nuggets are classified as pre-prepared frozen meals. These products are convenient to consumers as they are easy to prepare and allow for long storage by freezing. Over the years, spoilage of frozen food products caused by fungi has been a continual problem for the food industry since mold can develop when frozen foods are allowed to attain temperatures of − 10 °C, or above. The growth of fungi on the food surface results in economic losses and represents a hazard to public health due to the possibility of mycotoxin production. The aim of this study was to identify the species of filamentous fungi involved in the spoilage of frozen chicken nuggets and determine their ability to produce mycotoxins under laboratorial conditions. A total of 7 samples of frozen chicken nuggets were analyzed by dilution plating in potato dextrose agar (PDA). These products had been returned by customers due to visible mold growth on their surface. The predominant species found were Penicillium glabrum, Penicillium polonicum, Penicillium manginii, Penicillium crustosum, Penicillium commune, and Penicillium solitum. Analysis of the profile of secondary metabolites was carried out in HPLC after growing the isolates in Czapek yeast autolysate agar (CYA) and yeast extract agar and sucrose (YESA) and extracting the extrolites with a solution of ethyl acetate, dichloromethane, methanol, and formic acid. Some isolates of these species showed an ability to synthesize mycotoxins such as cyclopiazonic acid citreoviridin, roquefortine C, penitrem A, and verrucosidin under standard conditions. Considering the occurrence of fungal spoilage in frozen food and the potential hazard involved, more studies on psychrophilic fungi growth in foods stored at low temperatures are necessary.     

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.     

Mould growth on the Norwegian semi-hard cheeses Norvegia and Jarlsberg

Visible moulds were isolated and identified from 102 samples of each of the Norwegian types of semi-hard cheeses called Norvegia and Jarlsberg. Penicillium species made up 98.1 and 89.2% of the isolates from the Jarlsberg and Norvegia cheeses, respectively. The most frequently occurring species on both was P. roqueforti subspecies roqueforti. The four species Penicillium roqueforti subspecies roqueforti, P. commune, P. palitans and P. solitum made up 69.8% of the total number of isolates from the Norvegia cheese and 81.0% of the total number of isolates from the Jarlsberg cheese.     

Use of ozone to reduce molds in a cheese ripening room

Cheese ripening rooms have an unusual environment, an environment that encourages mold growth. Ozone has been applied in various ways in the food industry. One useful advantage of ozone is that it inactivates molds. In this study, a cheese ripening room was ozonated, and the effectiveness of this treatment was evaluated both in air and on surfaces through sampling on a weekly basis over a 3-month period. The results obtained indicate that ozone treatment reduced the viable airborne mold load but did not affect viable mold on surfaces. Only by wiping the surfaces with a commercial sanitizer was it possible to decrease the viable mold load on surfaces. To improve overall hygiene in the ripening room, a combination of cleaning regimes is recommended. The mold genera occurring most frequently in the air of the cheese ripening room were Penicillium, Cladosporium, and Aspergillus, which accounted for 89.9% of the mold isolates. Penicillium and Aspergillus were identified to the species level, and data showed that P. brevicompactum and P. aurantiogriseum, as well as A. versicolor, were the species most frequently isolated.     

Identification of fungal contamination and determination of mycotoxigenic molds by micellar electrokinetic capillary chromatography in smoked paprika

The purpose of this work was to analyze the fungal contamination in smoked and unsmoked paprika processed from different cultivars of pepper and to investigate the ability of these and other mycotoxigenic molds to grow and synthesize mycotoxins in smoked paprika. Eighteen mycotoxins were evaluated using micellar electrokinetic capillary chromatography. No relevant differences were found in fungal contamination between smoked and unsmoked paprika. The number of yeasts obtained was low, ranging from 0.4 to 3.29 log CFU g(-1); most of the yeast strains were identified as Cryptococcus spp. followed by Candida spp. All mold counts were <4 log CFU g(-1). Aspergillus, Cladosporium, Penicillium, and Fusarium were the predominant hyphomycete genera. Six mycotoxins were identified in the extracts of several strains isolated from paprika and incubated on malt extract agar. Penicillium expansum followed by Penicillium citrinum and Penicillium raistrickii were the dominant mycotoxigenic fungi isolated. Most of themycotoxin-producing fungi produced detectable amounts of mycotoxins when grown on paprika agar.     

Mycotoxin production capability of Penicillium roqueforti in strains isolated from mould-ripened traditional Turkish civil cheese

Mould-ripened civil is a traditional cheese produced mainly in eastern Turkey. The cheese is produced with a mixture of civil and whey curd cheeses (lor). This mixture is pressed into goat skins or plastic bags and is ripened for more than three months. Naturally occurring moulds grow on the surface and inside of the cheese during ripening. In this research, 140 Penicillium roqueforti strains were isolated from 41 samples of mould-ripened civil cheese collected from Erzurum and around towns in eastern Turkey. All strains were capable of mycotoxin production and were analysed using an HPLC method. It was established that all the strains (albeit at very low levels) produced roquefortine C, penicillic acid, mycophenolic acid and patulin. The amounts of toxins were in the ranges 0.4–47.0, 0.2–43.6, 0.1–23.1 and 0.1–2.3 mg kg^?1, respectively. Patulin levels of the samples were lower than the others. The lowest level and highest total mycotoxin levels were determined as 1.2 and 70.1 mg kg^?1 respectively. The results of this preliminary study may help in the choice of secondary cultures for mould-ripened civil cheese and other mould-ripened cheeses.     

Enzyme-linked immunosorbent assay for detection of molds in cheese and yogurt

An enzyme-linked immunosorbent assay was developed for the detection of molds in dairy products. New Zealand White female rabbits were immunized with .45 mg of partially purified extracellular antigen from freeze-dried culture filtrates of Aspergillus versicolor, Cladosporium herbarum, Geotrichum candidum, Mucor circinelloides, and Penicillium chrysogenum. Blood was drawn at various intervals, and antibodies were separated and purified. Antibody-peroxidase conjugates were prepared with the following ratios being the optimum ones: A. versicolor 10:20; C. herbarum 5:10; G. candidum 1:10; M. circinelloides 5:5; and P. chrysogenum 10:10. The assays were sensitive within a range of 1 ng to 1 microgram/ml, depending on the mold used. Inhibition tests were done for each mold with concentrations of 0 to 5000 micrograms/ml of antigen. The enzyme-linked immunosorbent assay tests for Cladosporium, Geotrichum, and Mucor were only inhibited by antigens from other species of the same genus; whereas there was crossreaction between antibodies and antigens of species of Penicillium and of Aspergillus. Citrate buffer was best for extracting the mold from cheese and yogurt. The extract was adjusted to pH 7.2 and ELISA was performed. Results showed that these molds can be detected in Cheddar and cottage cheeses and yogurt within 2 d, which is before mold growth is visible in these products.     

应用DGGE技术研究玉米储藏过程中的霉菌群落时空分布特征

研究储粮过程中霉菌污染的发生规律,旨在减少霉菌及真菌毒素的污染,提高我国粮食安全。利用变性梯度凝胶电泳研究粮库中不同储藏时间和空间的玉米样品中霉菌群落的特点。结果表明,本研究中储藏玉米的霉菌污染以青霉、曲霉和毛霉为主,其霉菌群落的变化与储藏时间具有较强的相关性,而与其在粮库中的空间位置相关性较小。在储藏时间方面,储藏1a和3a的样品中霉菌群落具有较大的差异,而储藏2a样品的霉菌群落处于过渡期状态。本实验探究储藏玉米中霉菌群落的时空分布特征,可以为建立准确可靠的霉菌群落模型提供理论支持和数据支持。     

Mould contaminants on Jarlsberg and Norvegia cheese blocks from four factories.

Visible mould from 225 blocks of the Norwegian semi-hard cheeses Jarlsberg and Norvegia from four factories were subcultured and identified. Altogether 23 different fungal species were detected. The two most important contaminating species were Penicillium commune and P. palitans, constituting 21.4% and 17.9% of the total isolates, respectively. The other dominating contaminants were P. roqueforit spp. roqueforti, Geotrichum candidum, P. solitum and P. crustosum. These species, together with P. commune and P. palitans, represented 80.9% of the total isolates. P. commune, P. palitans, P. roqueforti spp. roqueforti and P. solitum were most common contaminants on cheese produced in all four factories, while G. candidum was found to be important on Jarlsberg cheese from only one factory. P. crustosum was one of the dominating species on Norvegia cheese.