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Mould Spoilage of Foods and Beverages: Using the Right Methodology Food Microbiology 81 (2019) 51e62 Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm Mould spoilage of foods and beverages: Using the right methodology * Emilia Rico-Munoz a, , Robert A. Samson b, Jos Houbraken b a BCN Research Laboratories, Inc., 2491 Stock Creek Blvd., Rockford, TN 37853, USA b Westerdijk Fungal Biodiversity Institute, Dept. Applied and Industrial Mycology, Uppsalalaan 8, Utrecht, CT 3584, The Netherlands article info abstract Article history: Fungal spoilage of products manufactured by the food and beverage industry imposes significant annual Received 5 December 2017 global revenue losses. Mould spoilage can also be a food safety issue due to the production of mycotoxins Received in revised form by these moulds. To prevent mould spoilage, it is essential that the associated mycobiota be adequately 16 March 2018 isolated and accurately identified. The main fungal groups associated with spoilage are the xerophilic, Accepted 29 March 2018 heat-resistant, preservative-resistant, anaerobic and psychrophilic fungi. To assess mould spoilage, the Available online 30 March 2018 appropriate methodology and media must be used. While classic mycological detection methods can detect a broad range of fungi using well validated protocols, they are time consuming and results can Keywords: Mould spoilage take days or even weeks. New molecular detection methods are faster but require good DNA isolation Mould detection techniques, expensive equipment and may detect viable and non-viable fungi that probably will not spoil Xerophilic fungi a specific product. Although there is no complete and easy method for the detection of fungi in food it is Heat-resistant fungi important to be aware of the limitation of the methodology. More research is needed on the develop- Preservative-resistant fungi ment of methods of detection and identification that are both faster and highly sensitive. © 2018 Elsevier Ltd. All rights reserved. Contents 1. Introduction . ................................................. 52 2. Conventional methodology for the detection and enumeration of spoilage fungi . ..................... 52 2.1. Xerophilic fungi . ........................52 2.1.1. Moderate xerophiles . ........................53 2.1.2. Extreme xerophiles . ........................53 2.2. Heat-resistant fungi . ........................54 2.3. Preservative-resistant fungi . ........................55 2.4. Anaerobic fungi . ........................55 2.5. Psychrophilic and psychrotolerant fungi . ........................56 3. Molecular detection methodology . ............................................ 57 3.1. DNA extraction . ........................57 3.2. PCR and qPCR methods . ........................57 3.3. Next generation sequencing . ........................58 4. Identification and typing . ................................................. 58 4.1. Identification . ........................58 4.1.1. Sequence based identification . ........................58 4.1.2. MALDI-TOF MS . ........................59 4.2. Strain typing . ........................59 4.2.1. From multilocus sequence analysis to whole genome sequence typing . ........................59 4.2.2. AFLP............................................................. ............................ ........................59 4.2.3. Microsatellite analysis . ........................59 5. Conclusions . ................................................. 59 * Corresponding author. E-mail address: [email protected] (E. Rico-Munoz). https://doi.org/10.1016/j.fm.2018.03.016 0740-0020/© 2018 Elsevier Ltd. All rights reserved. 52 E. Rico-Munoz et al. / Food Microbiology 81 (2019) 51e62 Declaration of interest . ..................... 60 Conflicts of interest . ..................... 60 Acknowledgment and Funding Sources . ........................................60 References . ...................................................60 1. Introduction associated mycobiota be adequately isolated and accurately iden- tified. The appropriate methodology and media must be used. The Fungal spoilage of foods and beverages imposes significant high-water activity media such as malt extract agar (MEA) or annual global revenue losses for the food and beverage industries. dichloran rose bengal chloramphenicol (DRBC) agar used for spe- Mould spoilage can also be a food safety issue due to the production cies which grow at high water activity will not be able to recover of mycotoxins or allergens by these moulds. To avoid or reduce many of the moderate xerophilic moulds due to growth competi- mould spoilage, several hurdles can be used: (1) reducing the water tion by moulds that grow faster at these water activities (Hocking activity of the food, (2) thermal processing, (3) addition of pre- and Pitt, 1980; Hocking, 1992). Extreme xerophiles such as Xero- servatives, (4) reduction of oxygen in the packaging using vacuum, myces bisporus need media containing at least 50% glucose (Pitt and oxygen scavengers or modified atmosphere packaging (MAP), and Hocking, 2009). To isolate heat-resistant moulds from ingredients (5) refrigerated storage. However, the addition of each of these and environmental samples, a heat-shock step must be used (Rico- hurdles will be selecting for a different group of spoilage fungi Munoz et al., 2015). Preservative-resistant fungi can be isolated (Fig. 1). The use of two or more hurdles will reduce the number of using a selective media containing preservatives such as acetic acid moulds that can spoil the product. This is called the associated (Pitt and Hocking, 2009). mycobiota, typically comprised of several mould species, many of While classic mycological detection methods can detect a broad which are known to promote accelerated product spoilage range of fungi using well validated protocols, they are time (Filtenborg et al. 2004). The fungi groups associated with fungal consuming, required skilled personnel and some of the methods spoilage are: (1) xerophilic fungi that can grow at water activities have low sensitivity. New molecular detection methods are faster (aw) below 0.85 and spoil low and intermediate aw foods; (2) heat- but require good DNA isolation techniques, expensive equipment resistant fungi that survive pasteurization and baking, spoiling and may detect fungi that would not spoil a specific product. For the heat-processed products; (3) preservative-resistant fungi that can identification and other characterization of isolates, reference data grow in the presence of preservatives such as sorbates or benzoates (sequences, profiles) are needed and those are sometimes not and spoil beverages and foods that are preserved; (4) anaerobic available. Although there is no complete and easy method for the fungi that can grow under very low oxygen concentration and spoil detection of fungi in food it is important to be aware of the limi- vacuum or modified atmosphere packaged foods and (5) psychro- tation of the methodology. philic and psychrotolerant fungi that can grow under refrigeration This overview emphasizes the importance of using the right and spoil refrigerated foods and beverages. A few heat-resistant methodology for the detection, enumeration, and prevention of fungi such as Paecilomyces variotii are also preservative-resistant. potential fungal spoilage of food and beverage products. It also Some of the preservative-resistant moulds such as Penicillium compares classic to modern methodology for the detection of roqueforti can grow at low oxygen concentrations as well as under spoilage fungi. refrigeration. To prevent mould spoilage, it is essential that the 2. Conventional methodology for the detection and enumeration of spoilage fungi Conventional or classic methods have the advantage of detect- ing a wide variety of fungi using conventional media and meth- odology that have been validated and recognized as standard methods. There are methods specific for each group of spoilage fungi. Samson et al. (2004b, 2010) described the most common methods for the detection, isolation and characterization of food- borne fungi. In the case of direct microscopical examination of fungi growing on spoiled foods, a minimally trained technician can determine the fungal genus in a short time. However, most classical methods are time consuming requiring at least 5 day as well as trained laboratory technicians. To investigate food and beverage mould spoilage cases, the right methodology and media must be used. In this section, the right methodology used for the detection of xerophilic, heat-resistant, preservative-resistant, anaerobic and psychrophilic fungi will be described. 2.1. Xerophilic fungi Fig. 1. To avoid or reduce mould spoilage, several hurdles can be used: (1) reducing the water activity of the food, (2) thermal processing, (3) addition of preservatives, (4) Xerophilic fungi are defined by the ability to grow under con- reduction of oxygen in the packaging using vacuum, oxygen scavengers or modified ditions of reduced water activity, i.e. to complete their life cycles on atmosphere packaging (MAP), and (5) refrigerated storage. However, the addition of each of these hurdles will be selecting for a different group of spoilage fungi – the substrates that have been dried or concentrated, in the presence of associated mycobiota. high levels of soluble solids such as salts or sugars (Pitt and E. Rico-Munoz et al. / Food Microbiology 81 (2019) 51e62 53 Hocking, 2009). They can be subdivided among two groups per plate, spread with a sterile,
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