Characterization of molds isolated from smoked paprika by PCR-RFLP and micellar electrokinetic capillary electrophoresis

Molds are common contaminants of paprika meat products. The drying and storage stages of paprika processing are critical because they can provide molds with the conditions particularly appropriate for their growth and proliferation. Thus, an efficient and accurate characterization of the toxigenic molds of paprika is necessary. An RFLP analysis of the rRNA genes was performed by using a TaqI restriction enzyme. In addition, a micellar electrokinetic capillary chromatography (MECC) method was tested to analyze secondary metabolites produced by mold strains commonly found in paprika. This study was confirmed with a 5.8S-ITS region sequence analysis. A total of 31 isolates were identified by RFLP and MECC analysis. These showed stable RFLP profiles that were clearly different for the different genera and species, and were grouped into clusters together with the profiles of the 16 reference strains. MECC analysis provided additional characteristic peak patterns for the characterization of the mold species present. The characterized isolates were species of the genera Fusarium spp., Aspergillus spp., Penicillium spp., Cladosporium spp., Mucor spp. and Phlebia spp. The identifications were confirmed by the 5.8S-ITS region sequence analysis and by a BLAST search of the GenBank database. RFLP patterns with TaqI restriction enzyme and MECC profiles, either singly or combined, could be of great interest to distinguish molds in paprika.     

Production of secondary metabolites by some terverticillate penicillia on carbohydrate-rich and meat substrates

Most terverticillate penicillia isolated from dry-cured meat products are toxigenic, but their ability to produce hazardous metabolites on meat-based substrates is not well known. The production of extrolites by selected terverticillate penicillia isolated from dry-cured ham has been studied on carbohydrate-rich media (malt extract agar, Czapek yeast autolysate agar, rice extract agar, and rice), meat extract triolein salt agar, and ham slices. Chloroform extracts from the selected strains grown on malt extract agar were toxic for the brine shrimp (Artemia salina) larvae and VERO cells at a concentration of 2 mg/ml, but 0.02 mg/ml produced no toxic effect. Analysis by high-pressure liquid chromatography (HPLC) coupled with photodiode array detection (DAD) or with mass spectrometry (MS) and an atmospheric pressure chemical ionization (APCI) source revealed different biologically active metabolites: cyclopiazonic acid and rugulovasine A from Penicillium commune; verrucosidin, anacine, puberuline, verrucofortine, and viridicatols from Penicillium polonicum; arisugacin and viridicatols from Penicillium echinulatum; and compactin and viridicatols from Penicillium solitum. Most of these metabolites, including the amino acid-derived compounds, were produced in the media containing high levels of carbohydrates. High concentrations of nitrogen compounds in the medium does not imply a greater production of the metabolites studied, not even those derived from the amino acids. However, molds growing on dry-cured ham are able to synthesize limited amounts of some secondary metabolites, a fact not previously reported. The combination of HPLC coupled with DAD and MS-APCI was useful for identification of closely related terverticillate Penicillium species from dry-cured ham. These techniques could be used to characterize the risk associated with the potential production of secondary metabolites in cured meats.     

Proteolytic activity of lactic acid bacteria strains and fungal biota for potential use as starter cultures in dry-cured ham

During the processing of dry-cured meat products, sarcoplasmic and myofibrillar proteins undergo proteolysis, which has a marked effect on product flavor. Microbial proteolytic activity is due to the action of mostly lactic acid bacteria (LAB) and to a lesser extent micrococci. The proteolytic capacity of molds in various meat products is of interest to meat processors in the Mediterranean area. Eleven LAB and mold strains from different commercial origins were tested for proteolytic activity against pork myosin, with a view to possible use of these strains as starter cultures for Iberian dry-cured ham. Proteolytic activity was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The LAB strains with the highest proteolytic activity were Lactobacillus plantarum (L115), Pediococcus pentosaceus (Saga P TM), and Lactobacillus acidophilus (FARGO 606 TM). The best fungal candidate was Penicillium nalgiovense LEM 50I followed by Penicillium digitatum, Debaryomyces hansenii, and Penicillium chrysogenum.     

DNA typing methods for differentiation of yeasts related to dry-cured meat products

RFLP analysis of the ITS and 18S rDNA, RAPD-PCR using mini- and microsatellite primers and RFLP analysis of mitochondrial DNA were examined to discriminate yeasts related to dry-cured meat products at species and strain level. Seven species and 35 strains of yeasts usually found in dry-cured meat products were tested. RFLP analysis of the ITS1-5.8S rDNA-ITS2 and 18S rDNA did not allow the separation at species level of all of the species tested. RAPD with a M13 primer was found to be useful for differentiation of Rhodotorula mucilaginosa, Candida zeylanoides, Yarrowia lipolytica, Debaryomyces hansenii and Saccharomyces cerevisiae. However, no differences were observed between Debaryomyces polymorphus and Pichia carsonii. RAPD analysis with microsatellite primers (GACA)(4), (GTG)(5) and (GAC)(5) enabled discrimination at species and strain level. However, the degree of discrimination by means of RAPD-PCR depends highly on the primers used. Thus, the PCR fingerprinting with primer (GACA)(4) enabled a higher level of discrimination than primers (GAC)(5) and (GTG)(5). The RFLP analysis of mtDNA allowed the discrimination at the species and strain level except for R. mucilaginosa, where no polymorphisms were observed in the strains tested. RAPD analysis with primer (GACA)(4) and the restriction analysis of mtDNA used in the present work are useful for the differentiation at species and strain level of yeasts related to dry-cured meat products.     

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.     

干腌火腿中霉菌毒素研究的进展

从干腌火腿中分离到一些霉菌菌株在培养基中生长时可以产生黄曲霉毒素、环匹阿尼酸、青霉酸、梗曲霉素、展青霉素、灰黄霉素、霉酚酸、赭曲霉毒素、橘青霉素和疣孢苷啶等多种霉菌毒素。生成霉菌毒素的霉菌都属于青酶和曲霉。鲜绿青霉可以在干腌火腿中生成环匹阿尼酸,且这种霉菌毒素在干腌火腿中具有较高的稳定性,从而对干腌火腿的安全性构成很大的威胁。将无毒性的霉菌菌株进行培养并接种到干腌火腿上,可防止干腌火腿中形成霉菌毒素。     

Potentially toxigenic fungi from selected grains and grain products

A total of 85 grain and grain product samples (including corn meal, corn muffin mix, popcorn, various types of rice, and self‐rising, all‐purpose unbleached and whole wheat flour) from U.S. retail were tested for fungal contamination levels and profiles using conventional plating as well as molecular methods. The results of this study showed that over 90% of wheat flour and corn product samples and 73% of rice samples tested carried live fungi. Popcorn carried the highest fungal levels reaching 5.45 log₁₀ colony forming units (cfu) per gram followed by corn meal (reaching 5.38 log₁₀ cfu/g). Mold and yeast counts in rice and wheat flour reached 3.30 log₁₀ and 3.28 log₁₀ cfu/g, respectively. The predominant molds in wheat flour were aspergilli and fusaria found in 50 and 46% of samples, respectively; Fusarium spp. were the most frequent contaminants of corn‐based products found in 74% of the samples followed by penicillia (present in 44% of tested samples). Rice, conversely, contained mainly Aspergillus, Fusarium, and yeasts (each found in 21% of the samples).

Practical applications

Toxigenic molds are often contaminating stored grains and grain products and under improper storage conditions could cause spoilage of these commodities accompanied with production of toxic secondary metabolites, mycotoxins. Mycotoxins are known to cause illnesses in humans and animals. Therefore, monitoring the presence and inhibiting the growth of these organisms is critical for achieving and maintaining high quality products, suitable for human and animal consumption, and free of health hazards. Establishing toxigenic mold profiles in stored grains and their derivatives can point to correct storage management and thus reduction/elimination of spoilage and mycotoxin production in these products. In this study we tested several corn, rice, and wheat flour commodities for live potentially toxigenic fungal species. Our findings can help select proper storage management techniques for these commodities.     

腌腊肉制品中真菌毒素污染现状及防控研究进展

腌腊肉制品在加工和贮藏过程中容易受到真菌的污染,部分丝状真菌在一定条件下能够代谢产生真菌毒素,使肉制品存在毒素污染的安全风险,严重威胁人体健康。本文对腌腊肉制品真菌毒素的来源、种类（包括黄曲霉毒素、赭曲霉毒素A、桔青霉素和环匹阿尼酸等）及危害进行了简述,调查了国内外腌腊肉制品真菌毒素污染的现状,并对腌腊肉制品中真菌毒素的防控措施进行了综述,以期为腌腊肉制品的安全生产提供参考。     

FUSARIUM MOLDS AND THEIR MYCOTOXINS

An overview is presented covering the occurrence of Fusarium species in raw food and feed materials and the mycotoxins produced by these molds. A study of the literature (reported from 1969 until now) revealed the occurrence of over 60 Fusarium species in these raw materials. It appears that the most reported species occur worldwide and in a large variety of raw food products, such as cereals, vegetables and fruits. These Fusarium species occurring in samples are producers of toxigenic secondary metabolites of which more than 100 have been described. Most of them are referred to as mycotoxins of which trichothecencs (e.g., deoxynivalenol, nivalenol, T-2 toxin, diacetoxyscirpenol), fumonisins and zearalenones are most frequently reported .     

Is ergosterol a new microbiological quality parameter in foods or not?

One of the most important spoilage factors of foods is molds. Therefore, it is important to determine the presence of mold in foods quickly because of the deterioration of aroma, flavor, appearance, and textural structure as well as the mycotoxins, which are toxic secondary metabolites of molds. Twenty-five percent of agricultural products worldwide are infected with mycotoxins directly or indirectly. With the global population increasingly rising, the need for access of safe and adequate food in the future has great importance. Quantification of ergosterol, a constituent of the membrane of molds and a precursor of vitamin D₂, is a feasible method for determination of fungal contamination in foods. The aim of this review is to discuss the possibility of using ergosterol as an indicator of mold growth in foods.     

Diversity and toxigenicity among members of the Bacillus cereus group

Members of the Bacillus cereus group were isolated from rice products by centrifugation-plating and conventional spread-plating methods. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) results showed broad diversity among the strains and revealed some associations among isolates from raw and cooked rice samples, at the genotypic level. A comparatively greater diversity among strains was observed in isolates from raw rice than those from cooked rice and, generally, the RAPD profiles of isolates from raw and cooked rice were different, with only a few of them common to both types of rice. The toxigenic potential of the isolates was also determined by molecular and immunoassay analyses. The results revealed that most isolates from the B. cereus group were potentially or actually toxigenic, and some isolates could produce both diarrhoeal and emetic toxins. Generally, isolates belonging to the B. cereus group with the same RAPD pattern were shown to have a similar profile of enterotoxigenicity.     

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.     

Characterization of Penicillium roqueforti strains used as cheese starter cultures by RAPD typing

Seventy-six strains of Penicillium roqueforti used as starter cultures for mould ripened blue cheeses have been analysed for their RAPD genotype by using three different primers. A comparison of the RAPD patterns within each primer group revealed that the genetic constitution of the strains was similar, as most of the strains showed very similar overall patterns. Despite these similarities with each primer, distinct RAPD genotype groups could be identified. With one of the primers, it was possible to detect two heteropolymorphic DNA regions resulting in 13 different groups. With the other two primers, three or four groups could be identified. Between the groups of the different primers marked correspondence with respect to strain distribution could be observed, indicating that the polymorphisms detected by the primers were not independent. The RAPD patterns were compared to the production of secondary metabolites. A correlation was observed between the RAPD patterns of all primers and the production of mycophenolic acid. In addition, one of the primer (ari1) was able to distinguish between P. roqueforti strains producing larger or smaller numbers of metabolites.     

Development of a PCR protocol to detect aflatoxigenic molds in food products

Aflatoxins are secondary metabolites produced mainly by Aspergillus species growing in foodstuffs. Because aflatoxins have important health effects, the detection of early contamination of foods by aflatoxigenic molds should be useful. In the present work, a reliable conventional PCR method for detecting aflatoxigenic molds of various species was developed. Fifty-six aflatoxigenic and nonaflatoxigenic strains commonly reported in foodstuffs were tested. Aflatoxin production was first confirmed by micellar electrokinetic capillary electrophoresis or/and high-pressure liquid chromatography-mass spectrometry. Based on the conserved regions of the O-methyltransferase gene (omt-1) involved in the aflatoxin biosynthetic pathway, six primer pairs were designed. With only the designed primer pair AFF1-AFR3, the expected PCR product (381 bp) was obtained in all of the tested aflatoxigenic strains of various species and genera. Amplification products were not obtained with this primer pair for any of the nonaflatoxigenic reference molds. However, an amplicon of 453 bp was obtained for all aflatoxigenic and nonaflatoxigenic mold reference strains with a PCR protocol based on the constitutive fungal β-tubulin gene, which was used as a positive fungal control. The PCR protocol based on omt-1 detected as little as 15 pg of DNA from aflatoxigenic molds and 10(2) to 10(3) CFU/g in contaminated food samples. This PCR protocol should be used as a routine technique to detect aflatoxigenic molds in foods.     

Exo-metabolites of mycelial fungi isolated in production premises of cheese-making and meat-processing plants

Data were obtained on the species composition of mycelial fungi isolated from the air of workrooms and production premises in cheese-making and meat-processing plants. The strains studied were shown to be capable of producing various low molecular weight compounds. Many of them are mycotoxins such as α-cyclopiazonic acid (CPA), mycophenolic acid (MPA), citrinin, cladosporin, roquefortine and ergot alkaloids. The profiles of the secondary metabolites were used to elucidate the species’ names of the isolated strains.     

Selection of antifungal protein-producing molds from dry-cured meat products

To control unwanted molds in dry-cured meats it is necessary to allow the fungal development essential for the desired characteristics of the final product. Molds producing antifungal proteins could be useful to prevent hazards due to the growth of mycotoxigenic molds. The objective has been to select Penicillium spp. that produce antifungal proteins against toxigenic molds. To obtain strains adapted to these products, molds were isolated from dry-cured ham. A first screening with 281 isolates by the radial inhibition assay revealed that 166 were active against some of the toxigenic P. echinulatum, P. commune, and Aspergillus niger used as reference molds. The activity of different extracts from cultured medium was evaluated by a microspectroscopic assay. Molds producing active chloroform extracts were eliminated from further consideration. A total of 16 Penicillium isolates were screened for antifungal activity from both cell-free media and the aqueous residues obtained after chloroform extraction. The cell-free media of 10 isolates that produced a strong inhibition of the three reference molds were fractionated by FPLC on a cationic column. For protein purification, the fractions of the three molds that showed high inhibitory activity were further chromatographed on a gel filtration column, and the subfractions containing the highest absorbance peaks were assayed against the most sensitive reference molds. One subfraction each from strains AS51D and RP42C from Penicillium chrysogenum confirmed the inhibitory activity against the reference molds. SDS-PAGE revealed a single band from each subfraction, with estimated molecular masses of 37 kDa for AS51D and 9 kDa for RP42C. Although further characterisation is required, both these proteins and the producing strains can be of interest to control unwanted molds on foods.     

Generation of non-protein nitrogen and volatile compounds by Penicillium chrysogenum Pg222 activity on pork myofibrillar proteins

A non-toxigenic strain of proteolytic Penicillium chrysogenum Pg222 isolated from dry-cured ham was tested for its ability to generate non-protein nitrogen (NPN) and volatile compounds from muscle myofibrillar proteins. The activity of mold led to higher accumulation of non-protein and amino acidic nitrogens than control samples. Volatile compound analysis revealed the presence of branched compounds, such as 3-methylbutanal and 3-methylbutanol only in samples inoculated with the mold. Similarly, compounds such as ethanol, propanol and 2-methoxy ethanol were detected only in inoculated samples at all sampling time. 3- and 2-methylpentane, benzoic and acetic acids, 2-butanone and 2-ethylhexanol, pyridine and 3-carene were detected occasionally, but only in the Pg222 batch. The proteolytic activity of P. chrysogenum Pg222 lead to accumulation of soluble NPN compounds, in addition to the generation of volatile compounds of great interest for dry-cured meat products. Therefore, this mold could be appropriate to be used as a non-toxigenic starter culture during the ripening of dry-cured meat products to stimulate proteolysis and flavour development.     

IDENTIFICATION OF HAZARDOUS FUSARIUM SECONDARY METABOLITES OCCURRING IN FOOD RAW MATERIALS

Hazardous secondary metabolites produced by Fusarium molds were identified in the first step of a risk assessment procedure, the hazard identification. These mycotoxins are excreted, often simultaneously, by the fungus into plant tissue. A literature survey revealed the occurrence of 61 Fusarium species in agricultural produce (cereals, vegetables, fruits) of which 35 species were reported to produce a total of 137 secondary metabolites in laboratory experiments. This literature review shows that 41 (30%) of these secondary metabolites have been tested and found toxic in various assays and should be considered hazardous if present in food. Six of these were reported to be related to human intoxications. Four metabolites belonged to the trichothecenes (T-2 toxin, nivalenol, deoxynivalenol and acetyldeoxynivalenol), the others were zearalenone and fumonisin B₁. Twelve metabolites, including the previously mentioned six, were reported to have been tested in feeding trials using vertebrates. These twelve secondary metabolites should be regarded as most important and need to be evaluated in each risk assessment procedure for mycotoxins in food commodities .     

Microbiological evaluation and molecular characterization of bifidobacteria strains in commercial fermented milks

The aim of this study was to evaluate the presence of Bifidobacterium animalis subsp. lactis in commercial dairy products using different molecular techniques. We analyzed the microbiological composition of 13 commercial fermented milks available in the Spanish market. Thirteen strains of genus Bifidobacterium were isolated from these products and were identified by genus-specific PCR, by fluorescence in situ hybridization (FISH), by multiplex PCR and amplified ribosomal DNA restriction analysis (ARDRA). The same sets of strains were typed by randomly amplified polymorphic DNA (RAPD) analysis and by amplified fragment length polymorphism technique (AFLP). All strains were identified as B. animalis subsp. lactis using ARDRA and multiplex PCR techniques. Similarity between strains was evaluated based on RAPD and AFLP profiles. The isolated strains showed similar profiles by using these techniques, revealing the reduced genetic variability existing among commercial strains, and all these profiles were reproducible in repeated analysis. ARDRA and multiplex PCR are techniques that allow differentiation of the bifidobacteria at genus and species level, but do not indicate if they are different strains, for which reason the RAPD technique is very useful. All bifidobacteria isolated from commercial fermented milks in Spain belong to the same species B. animalis subsp. lactis. Our results demonstrate the necessity to control the presence of bifidobacteria in commercial fermented milks, not only at species level but also at strain level. Multiplex PCR and RAPDs are the most suitable, rapid and precise techniques to identify all bifidobacteria contained in fermented milk products at genus-, species-, and strain levels.     

Contribution of a selected fungal population to the volatile compounds on dry-cured ham

Dry-cured ham is obtained after several months of ripening. Different fungi strive on the surface, including toxigenic molds. Proteolysis and lipolysis by the endogenous and microbial enzymes seem to play a decisive role in the generation of flavor precursors in dry-cured meat products. In addition, fungi show a positive impact on the volatile compounds of ripened pork loins. However, the contribution of the fungal population to flavor formation in dry-cured ham remains unclear. One selected strain each of Penicillium chrysogenum and Debaryomyces hansenii was inoculated as starter cultures on dry-cured ham. Volatile compounds extracted by solid phase micro-extraction technique were analyzed by gas chromatography/mass spectrometry. A trained panel evaluated flavor and texture of fully ripened hams. The wild fungal population on non-inoculated control hams correlates with higher levels of short chain aliphatic carboxylic acids and their esters, branched carbonyls, branched alcohols, and some sulfur compounds, particularly at the outer muscle. Conversely, P. chrysogenum and D. hansenii seem to be responsible for higher levels of long chain aliphatic and branched hydrocarbons, furanones, long chain carboxylic acids and their esters. The very limited impact of P. chrysogenum on pyrazines in inoculated hams can be due to the activity of the yeast. Lower levels for some of the more volatile linear carbonyls at the ham surface suggest an anti-oxidant effect by micro-organisms. The differences in volatile compounds did not show a neat impact on flavor in the sensorial analysis. Nonetheless, inoculated hams got a better overall acceptability, which has to be attributed to their improved texture. The lower toughness of inoculated hams is a direct consequence of an early settling of a highly proteolytic mold. Thus, the use of selected fungi as starter cultures may be useful to obtain high-quality and safe dry-cured ham.