Abnormal Colorations of Mozzarella Cheese Caused by Phoma glomerata (Corda) Wollenw & Hochapfel

This paper describes an unusual type of abnormal coloration caused by the fungal species Phoma glomerata (P. glomerata) detected in samples of commercially available mozzarella cheese produced from cow’s milk. The presence of this fungus in dairy and cheese products has already been reported by other authors, along with other fungal contaminants; however, it has never been associated to specific alterations of cheeses. This is the first report of a macroscopic alteration of a soft cheese due to P. glomerata. Mozzarella cheese from four packages (two sealed and two already opened, three of which with evident macroscopic alterations) was analyzed by means of ISO methods for the detection of the main bacterial and fungal contaminants of cheese products. Culture tests carried out according to the ISO 21527-1:2008 method revealed presence of P. glomerata (from 1,100 CFU/g to 45,000 CFU/g). In addition, in both the previously opened packages, Acremonium spp. (100 CFU/g), Alternaria spp. (100 CFU/g), Pseudomonas fluorescens (25,000 CFU/g) and Pseudomonas putida (2,400 CFU/g) were also isolated. In sample N°4, contamination by P. glomerata was present, but in the absence of macroscopic changes. These results show that P. glomerata is able to contaminate mozzarella cheese, causing macroscopically visible alterations of the product; this may have serious consequences in terms of sales. With regard to the possible effects on human health, further studies are needed in order to assess the toxic effect of the fungus. As a result of the episode described, the Italian health authorities issued a RASFF (Rapid Alert System for Food and Feed) early warning notice, a key E.U. tool to ensure the cross-border flow of information in order to react swiftly when risks to public health are detected in the food chain.


Introduction
Mozzarella is a typical Italian cheese product made by stretching and kneading the drained curd mass.It is mainly made from cow's milk, but over recent years mozzarella made with buffalo's milk has becoming increasingly popular.There has been an increase in national demand and important commercial channel have also opened up both in Europe and toward third party countries, in particular the USA and Asia.
In recent years, color anomalies have been reported in fresh cheeses, and particularly in mozzarella cheeses (so-called "blue mozzarella"), caused by biotypes A and B of the species Psedomonas putida, Pseudomonas chlororaphis, Pseudomonas gessardii and Pseudomonas brassicacearum, which have been indicated as responsible for yellow/orange color alterations in cottage cheese (Giaccone, 2010) and mozzarella (Cantoni et al., 2006;Cantoni, Stella, Cozzi, Iacumin, & Comi, 2003;Soncini, Marchivio, & Cantoni, 1998).These reports have triggered health alerts in several European and non-European countries and have raised concerns among consumers, who are increasingly attentive to food safety.Indeed, in the case described here, the anomaly of the product was reported to the health authorities by consumers who had purchased the cheese through the regular channels.Cheeses are subject to numerous varieties of microbial and fungal contamination.In certain types of cheese, the growth of mold is desirable, as it enhances the organoleptic characteristics of the product.Some types of mold, however, cause unwanted changes that negatively affect the quality of the product, resulting in commercial and economic losses.In addition, some kinds of molds can produce mycotoxins, representing potential risks for human health.The genera most commonly responsible for such alterations are: Cladosporium (C.cladosporioides, C. herbarum), Penicillium (P.commune, P. glabrum) and Phoma (Hocking & Faedo, 1992).Phoma is the largest and most widespread genus of the order Pleosporales, which includes more than 2000 cosmopolitan species (Mold & Bacteria Consulting Laboratories 2005).Owing to its great ecological versatility, it is widespread in air, sea-water, soil and leaves, and can also be isolated from wood, paper, textiles (wool), leather and aquatic organisms (Dörr et al., 2011).Being a common airborne contaminant and allergen in indoor environments, Phoma can cause infections, Type-1 allergies (rhinitis, asthma), pneumonitis, keratitis and skin and subcutaneous lesions in humans.A wide variety of vegetables (Kocić-Tanackov et al., 2010), fresh fruits, meat, dairy and meat-derived products (Sørensen, Jacobsen, Nielsen, Frisvad, & Koch, 2008), and animal and vegetable fat (Samson, Hoekstra, & Frisvad, 2004) have occasionally been reported as contaminated by Phoma spp.In particular, the species Phoma glomerata (P.glomerata) has been found in raw cow's milk (Lavoie, Touchette, St-Gelais, & Labrie, 2012), hard cheeses (Fente-Sampayo et al., 1995;Hocking & Faedo, 1992) and semi-hard cheeses (Hoekstra, Van Der Horst, & Samson, 1998).In a study conducted on cheddar cheese (Basilico, Debasilico, Chiericatti, & Vinderola, 2001), P. glomerata accounted for 63% of the fungi isolated.Moreover, the indoor environments of food-production facilities have often proved to be contaminated by various species of environmental fungal genera, including Phoma; in such situations, foodstuffs may be contaminated during the phases of processing or wrapping.The present paper is the first to describe adulteration caused by P. glomerata in commercially available mozzarella cheeses produced from cow's milk.

Samples Analyzed
The matrix tested consisted of four packages of mozzarella cheese immersed in water.Each contained 125 g of cheese, flow-packed in modified-atmosphere packaging, belonging to the same production lot.The cheese had been produced in Italy from bovine milk, with the addition of salt, rennet and lactic ferments, and were purchased by a consumer from a food supermarket.On opening, the consumer noticed strange dark spots on the product and reported the matter to the health authorities, which sent the sample to Department of food microbiology Istituto Zooprofilattico Sperimentale del Mezzogiorno of Catanzaro, Italy, for laboratory investigation.Two packages had already been opened, but the product was whole and unconsumed (indicated as samples N°1 and N°2), while the other two (samples N°3 and N°4) were still intact and sealed.

Visual Inspection of Samples
The tests were performed on all samples in sterile conditions through the use of biological hood and sterile equipment, and stored in same conditions until the end of analysis.External and internal portions of the samples underwent visual and olfactory examination in order to detect the presence of alterations, abnormal odors and variations in consistency.

Microbiological Tests
The samples underwent ISO (International Organization for Standardization) testing; the microbiological parameters determined for each sample and the test methods used were as follows: survey of Salmonella spp.(UNI EN ISO 6579:2004) The tests were performed on all samples in sterile conditions through the use of biological hood and sterile equipment.From each sample, 25 g of the product was taken for Salmonella spp and Listeria monocytogenes detection, and 10 g for the other microbiological parameters.Following homogenization and preparation of scalar dilutions on a decimal basis for quantitative testing, this material was seeded in culture media and incubated at the respective temperatures.Subsequently, in addition to colony counting, the various bacterial colonies were identified by means of macroscopic observation of the smears subjected to Gram staining and through biochemical testing with an automated system (VITEK 2TM Compact, bioMérieux France).Similarly, the developed filamentous fungal colonies were counted and separated on the basis of their macro-morphology.For each group detected, representative strains were transferred into appropriate media for identification: PDA (Potato Dextrose Agar, medium composition: 200 g potato, 20 g dextrose, 15 g agar,1000 ml distilled water) and MEA (Malt Extract Agar, medium composition: 30 g malt extract, 15 g agar, 1000 ml distilled water).Identification was carried out both on the basis of the macro-morphology of the colony and on the direct microscopic observation of the reproductive structures and spores (staining with Amman's lactophenol/fuchsin lactophenol).Specific taxonomical keys were finally used for strain characterization (Sutton, 1980;Samson et al., 2004).The incubation times and temperatures used were those recommended for the methods of testing for each specific microbiological www.ccsen parameter,

Results
The presen strictly be considered a pathogen; rather, it is an opportunistic fungus that can be involved in mycosis after entering the human body by chance.Nevertheless, the tests conducted in this study highlighted its ability to cause organoleptic alterations in mozzarella cheeses, with inevitable serious commercial consequences.Moreover, these findings should help industrial quality-control systems to identify sources of contamination.As a result of the episode described in this paper, the health authorities issued an RASFF (Rapid Alert System for Food and Feed) early warning notice.