Lisa Vallone

Identification of Penicillium aurantiogriseum species with a method of polymerase chain reaction-restriction fragment length polymorphism

Abstract A polymerase chain reaction method, added of restriction fragment length polymorphism analysis, was utilised to identify moulds, isolated from foods, belonging to Penicillium aurantiogriseum species. Suitable restriction enzymes were found. This simple method could be useful to face mycotoxin problem, discriminating toxic from non-toxic strains.

Aflatoxins in Organic Milk and Dairy Products

The search for aflatoxin M1 through the various stages of production of fresh organic cheese has to fulfill two requirements: firstly, to evaluate the actual wholesomeness of this product (which is served in elementary school cafeterias throughout Milan school districts); and secondly, to assess the conversion factor of mycotoxin that may be carried over through organic milk used to make crescenza cheese. The established limit for this mycotoxin, whose toxic effects are widely recognized, is 0.05 mg per kg of milk (EC reg. N. 466/01). The limit for cheese established by Swiss legislation is 0.25 mg per kg of cheese. The present study refers to Swiss regulations in the absence of current EC legislation for cheese.

Determination of styrene content in gorgonzola PDO cheese by headspace solid phase micro-extraction (HS-SPME) and gas-chromatography mass-spectrometry (GC-MS).

Control of the composition of products that are intended for use as packaging material is essential, particularly when these products come into direct contact with food. It is well known that plastics are not inert and that their residual monomers, starting substances, and additives are able to migrate into the food they contact. Among plastics, styrene is a common compound found in many plastic containers that can also be produced by the oxidation of Penicillium roqueforti used in gorgonzola Protected Denomination of Origin cheese manufacturing. Therefore, solid-phase microextraction combined with gas chromatography/mass spectrometry was applied in the present work to determine the styrene content in packaged and unpackaged gorgonzola cheese samples to understand styrene migration phenomena from plastic containers.

A survey of the mycobiota associated with larvae of the black soldier fly (Hermetia illucens) reared for feed production

Feed security, feed quality and issues surrounding the safety of raw materials are always of interest to all livestock farmers, feed manufacturers and competent authorities. These concerns are even more important when alternative feed ingredients, new product developments and innovative feeding trends, like insect-meals, are considered. The black soldier fly (Hermetia illucens) is considered a good candidate to be used as feed ingredient for aquaculture and other farm animals, mainly as an alternative protein source. Data on transfer of contaminants from different substrates to the insects, as well as the possible occurrence of toxin-producing fungi in the gut of non-processed insects are very limited. Accordingly, we investigated the impact of the substrate/diet on the intestinal mycobiota of H. illucens larvae using culture-dependent approaches (microbiological analyses, molecular identification through the typing of isolates and the sequencing of the 26S rRNA D1/D2 domain) and amplicon-based next-generation sequencing (454 pyrosequencing). We fed five groups of H. illucens larvae at the third growing stage on two substrates: chicken feed and/or vegetable waste, provided at different timings. The obtained results indicated that Pichia was the most abundant genus associated with the larvae fed on vegetable waste, whereas Trichosporon, Rhodotorula and Geotrichum were the most abundant genera in the larvae fed on chicken feed only. Differences in the fungal communities were highlighted, suggesting that the type of substrate selects diverse yeast and mold genera, in particular vegetable waste is associated with a greater diversity of fungal species compared to chicken feed only. A further confirmation of the significant influence of diet on the mycobiota is the fact that no operational taxonomic unit common to all groups of larvae was detected. Finally, the killer phenotype of isolated yeasts was tested, showing the inhibitory activity of just one species against sensitive strains, out of the 11 tested species.

Composition, proteolysis, and volatile profile of Strachitunt cheese.

Strachitunt, a blue-veined Italian cheese, received the Protected Designation of Origin (PDO) label in 2014. Its unique technological feature is represented by the dual-curd method of production. Strachitunt is produced from raw bovine milk with or without the inoculation of natural starter cultures of lactic acid bacteria, and the addition of secondary cultures of mold spores is not permitted by the product specification. Physico-chemical properties, proteolysis, and volatile profile of Strachitunt were investigated in 10 cheese samples (ripened for 75 d) made throughout spring 2015 and provided by the main cheese maker. Overall, composition parameters showed a large variability among samples. Cheese was characterized by an acid paste (pH 5.46) and a lower extent of proteolysis compared with other blue-veined varieties. The main chemical groups of volatile organic compounds were alcohols and esters, whereas ketones represented only a minor component. The erratic adventitious contamination by mold spores of the cheese milk, the unique dual-curd method of cheese-making, and the large time variability between the piercing time and the end of ripening could be highlighted as the main causes of both the distinctive analytical fingerprint and the scarce standardization of this blue-veined cheese.

Secondary metabolites from Penicillium roqueforti, a starter for the production of Gorgonzola cheese

The presence of mold in food, although necessary for production, can involve the presence of secondary metabolites, which are sometimes toxic. Penicillium roqueforti is a common saprophytic fungus but it is also the essential fungus used in the production of Roquefort cheese and other varieties of blue cheese containing internal mold. The study was conducted on industrial batches of Penicillium roqueforti starters used in the production of the Gorgonzola cheese, with the aim to verify the production of secondary metabolites. Nine Penicillium roqueforti strains were tested. The presence of roquefortine C, PR toxin and mycophenolic acid was tested first in vitro, then on bread-like substrate and lastly in vivo in nine cheese samples produced with the same starters and ready to market. In vitro, only Penicillium out of nine produced roquefortine C, four starters showed mycophenolic acid production, while no significant amounts of PR toxin were detected. In the samples grown on bread-like substrate, Penicillium did not produce secondary metabolites, likewise with each cheese samples tested. To protect consumers’ health and safety, the presence of mycotoxins needs to be verified in food which is widely consumed, above all for products protected by the protected denomination of origin (DOP) label (i.e. a certificate guaranteeing the geographic origin of the product), such as Gorgonzola cheese.

Functional characterization of Lactobacillus plantarum ITEM 17215: A potential biocontrol agent of fungi with plant growth promoting traits, able to enhance the nutritional value of cereal products

In this work, we explored the potential of 25 Lactobacillus plantarum strains isolated from cereals and milk-based products, testing characteristics related to antifungal activity and to nutritional quality. The tested strains demonstrated interesting beneficial traits, such as the ability to utilize fructo-oligosaccharides, prebiotic substances that help probiotic microorganisms to grow in the human gut, and to reduce phytate, an antinutrient present in cereal sector. Regarding mould inhibition, we highlighted the ability of the strains to inhibit Penicillium roqueforti, Mucor circinelloides and mycotoxinogenic moulds associated with cereal grains as Aspergillus flavus, A. niger, Fusarium verticillioides. Moreover, a moderate reduction of the bioavailability of aflatoxin AFB1 was detected. The selected L. plantarum strain ITEM 17215, showed a strong inhibitory ability towards fungal growth and was able to produce 1,2-dihydroxybenzene, benzoic acid, p-hydroxyphenyllactic acid and 3-phenyllactic acid. The latter compound, already described as efficient antifungal inhibitor, was the most abundant and its concentration was further increased by adding phenylalanine and phenylpyruvic acid in the growth medium. The metabolites produced by strain ITEM 17215 could also be related to the ability of the strain to induce cereal germination and promote plant growth. This aspect, not yet investigated in L. plantarum, could have interesting applications in the agro-food sector.

Evaluation of antifungal effect of gaseous ozone in a meat processing plant

Ozone is already known as effective food/environmental disinfection agent, thanks to its oxidative action towards microbial cell components. Bactericidal effect of ozone is well documented, while data concerning its inhibitory activity towards fungi are still ambiguous. Our study aims to evaluate the antimicrobial activity of gaseous ozone towards potentially contaminant fungi in a meat production plant in real working conditions. M2 and M5S5 plates were inoculated with Aspergillus niger, Penicillium roqueforti, Mucor racemosus, Saccharomyces cerevisiae strains and positioned in a deboning room, where gaseous ozone was dispensed throughout the night (until a maximum concentration of 20 ppm). Nine different points were chosen, based on height and distance from the ozone dispenser. After the treatment, the presence of strains growth was evaluated. Gaseous ozone did not show any inhibitory activity against mould strains, as optimum growth during all the trials was observed. An appreciable and constant microbicidal effect against S. cerevisiae was evidenced, with a mean value of 2.8 Log reduction. Our results suggest the importance of the definition of environmental and technical use conditions in order to optimise the antimicrobial efficacy of ozone in real working situations in food industries.

Identification of Moulds Belonging to the Penicillium Genus using a Polymerase Chain Reaction (PCR-SSCP) Method Followed by 'Computer Assisted' Statistical Analysis of the Electrophoretic Patterns

F. Colombo*, L. Vallone, R. Capoferri and I. Dragoni Dipartimento di Scienze e T ecnologie Veterinarie per la Sicurezza Alimentare, Universita degli Studi di Milano, V ia A. Grasselli 7, 20137 Milano, Italy *Correspondence: Dipartimento di Scienze e T ecnologie Veterinarie per la Sicurezza Alimentare, Universita degli Studi di Milano, V ia A. Grasselli 7, 20137 Milano, Italy E-mail: fabio.colombo@unimi.it