A taxonomic and ecological overview of cheese fungi

Abstract

Cheese is made from milk by a succession of microbes (bacteria, yeasts and fungi) that determine the consistency and flavor of the cheese. Apart from the emblematic species, Penicillium camemberti and Penicillium roqueforti, cheese fungi are not well known. Here we present a taxonomic and phylogenetic overview of the most important filamentous cheese Ascomycota based on 133 isolates provided by the producers of cheese and cheese starter cultures and 97 isolates from culture collections. We checked the congruence of different gene genealogies to circumscribe cheese species and our results allow us to propose molecular targets for their identification. To study their phylogenetic affiliation, we used LSU rDNA and showed that cheese fungi are found in two classes, the Eurotiomycetes with Penicillium species (Eurotiales) and Sporendonema casei/Sphaerosporium equinum (Onygenales), and the Sordariomycetes with Scopulariopsis species (Microascales) and Fusarium domesticum (Hypocreales). Some of these fungi, such as, P. camemberti, F. domesticum, Scopulariopsis flava and S. casei, are only known from cheeses and are probably adapted to this particular habitat, which is extremely rich in protein and fat. Other cheese fungi are ubiquitous, such as, P. roqueforti, Scopulariopsis candida and Scopulariopsis fusca.

Introduction

Cheese making began about 8000 years ago (Fox et al., 2000) and has spread throughout the world; this spread has led to a remarkable diversity of cheeses that nowadays is estimated at more than 1000 (Irlinger and Mounier, 2011). Although cheeses may be categorized by the origin of their milk (e.g., goat, sheep or cow), the most usual classification of cheeses is based on their consistency: fresh and soft (e.g., Camembert, Maroilles or Munster), pressed and fresh (Saint-Nectaire, Tomme, Pyrénées), pressed and aged (Emmental, Gruyère, Comté), pulled-curd (Mozzarella cheese) and blue vein (Bleu d'Auvergne, Fourme d'Ambert, Roquefort). Cheese is important to the culture of all European countries, extending as it does from Spain and Italy to Germany, the low countries, and France which is undeniably the country with the most diverse cheese culture, producing cheese of all categories. In 2009, for example, France produced 1 692 369 t of cow cheeses, 93 764 t of goat cheeses and 57 328 t of sheep cheeses (Agreste, 2009).

Cheese production requires particular know-how to control the cultivation of the molds, yeasts and bacteria that are inoculated into the milk and that play a major role in obtaining the desired flavor, aroma, texture, color, aspect, and nutritive elements of the cheese (Beresford, 2001, Irlinger and Mounier, 2011). Bacteria are necessary to prepare the milk for fungal growth by acidification. Subsequent development of filamentous fungi occurs during cheese ripening and in most cheeses fungi are present only on the surface; the exception is blue cheeses, where the fungus also develops inside the blue “veins” of the cheese. In addition to fungal starters inoculated in the milk, indigenous molds of the ripening rooms may colonize the surface of cheeses. These opportunistic uncontrolled microorganisms are usually connected with undesirable effects such a negative impact on flavor and taste, discoloration that is unacceptable to most consumers, or, rarely, production of toxic secondary compounds (Lund et al., 1995, Lund et al., 2003). Most Penicillium species are known to produce mycotoxins under laboratory conditions even among inoculated species such as Penicillium roqueforti and Penicillium camemberti (Frisvad et al., 2004). Although no direct evidence of toxicity to humans has been reported for cyclopiazonic acid produced by P. camemberti, absence of toxin in cheeses cannot be taken for granted (Pitt, 2002). P. roqueforti produces several secondary metabolites that are toxic, but it appears that they represent a low public health risk, even when blue-veined cheeses are consumed daily (Teubler and Engels, 1983). PR toxin has been shown to be unstable in cheese (Chang et al., 1993).

The commercial availability of filamentous fungi selected for their use in cheese making has allowed producers to achieve more consistent flavor and taste, as well as a more uniform appearance, of their cheeses. Whereas large-scale factories use this constancy to satisfy consumers, small artisanal producers try to preserve flavor and other characteristics that mark the originality of their products. However, both types of producers are faced with considering food safety concerns stemming from any uncontrolled mycobiota. In 2007, the European Food Safety Authority (EFSA) commented on fungi used as an additive in food and feed production, pointing out the need to improve our knowledge concerning their accurate identification and ability to produce secondary metabolites, some of which may be toxic (EFSA, 2007). Here, we take a first step in this journey by researching the evolutionary relationships of fungi used to make cheese and then use that information to design a molecular method for their rapid and positive identification.

Historically, fungal taxonomy has not been an easy task, mainly due to the difficulty of observing discriminating phenotypical characters at the species level. Molecular biology has opened access to genomic characters and to new concepts for taxonomic studies. Fungal taxonomists now routinely used the concordance of different gene genealogies (GCPSR: Genealogical Concordance Phylogenetic Species Recognition criterion) to delimit species because it appears congruent with, and more finely discriminating than, morphologogical and interfertility species recognition criteria (e.g., Taylor et al., 2000, Koufopanou et al., 2001, Dettman et al., 2003, Pringle et al., 2005, Le Gac et al., 2007, Giraud et al., 2008, Giraud et al., 2010). After species delimitation using the GCPSR criterion, DNA barcode markers can be chosen for rapid species identification (see http://connect.barcodeoflife.net/group/fungi).

Some cheese fungi are already well known, such as the two emblematic species P. camemberti (Frisvad and Samson, 2004, Giraud et al., 2010) and P. roqueforti (Frisvad and Samson, 2004), however, others have received less attention and have no correct identification. In terms of evolution, it is also interesting to infer their phylogenetic affinities with saprophytic and pathogenic fungi.

This study aims 1) to identify correctly cheese filamentous ascomycetes, 2) to realize a rigorous circumscription of the species using the concordance of different gene genealogies, 3) to establish relationships between isolates of the cheese environment and isolates from other environments and 4) to discuss hypotheses about their adaptation to the dairy environment.