Food Control 41 (2014) 7e12 Contents lists available at ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont Risk analysis and rapid detection of the genus Thermoascus, food spoilage fungi Kouichi Hosoya a, Motokazu Nakayama a, Daisuke Tomiyama a, Tetsuhiro Matsuzawa b, Yumi Imanishi c, Seiichi Ueda d, Takashi Yaguchi b,* a Global R&D-Safety Science, Kao Corporation, 2606 Akabane, Ichikai-machi, Haga-Gun, Tochigi 321-3497, Japan b Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan c Department of Public Health, School of Medicine, Kurume University, 67 Asahimachi, Kurume, Fukuoka 830-0011, Japan d Graduate School of Human Health Science, University of Nagasaki, Japan article info abstract Article history: Recently the numbers of spoilage incidents in food industry by the species of Thermoascus are increasing, Received 30 August 2013 but the risk of food spoilage have never been evaluated. Received in revised form It became obvious that their heat-resistances were higher than those of other heat-resist fungi, 2 December 2013 Byssochlamys, Hamigera and Neosartorya by our analyses. On the other hand, Thermoascus aurantiacus Accepted 17 December 2013 and Byssochlamys verrucosa had the idh gene, but they showed no patulin production in Potato dextrose broth or Czapek-glucose medium. Therefore, Thermoascus must be discriminated from other fungi in the Keywords: food industry. We developed a rapid and highly-sensitive method of detecting Thermoascus in the genus D value fi Food spoilage level by using PCR. This method is expected to be extremely bene cial for the surveillance of raw ma- Heat-resistance terials in the food production process. idh gene Ó 2013 Elsevier Ltd. All rights reserved. PCR 1. Introduction a variety of agricultural products, including maize stored in sub- Sahara Africa and olive and olive cake in Morocco (Roussos et al., The history of food spoilage due to heat-resistant fungi dates 2006; Wareing, 1997), and in food-related environments (Ueda & back to the 1930s when incidents in canned foods were first re- Udagawa, 1983; Yaguchi, Someya, & Udagawa, 1995). Recently the ported; numerous incidents of spoilage of processed fruit juices and numbers of spoilage accidents in various processed tea and fruit beverages have been reported to date (Dijksterhuis, 2007; Kikoku, juice products by this genus are increasing (in our experience). In Tagashira, & Nakano, 2008; Samson, Hoekstra, Lund, Filtenborg, & addition, the production of high amounts of amylase and cellulase Frisvad, 2004; Tournas, 1994). It is known that asexual fungi have by Thermoascus spp. can markedly alter food product properties, hyphae and conidia that are susceptible to heat, and these fungi are and the high thermostability of these enzymes makes them diffi- typically killed with heat treatment at 70 C for 10 min. However, cult to inactivate with heat treatment (Adams, 1992; Feldman, heat-resistant fungi in genera Byssochlamys, Neosartorya, Hamigera, Lovett, & Tsao, 1988). However, the risk for food industry caused and Thermoascus belonging to the order Eurotiales (Plectomycetes) by this genus has never been reported, except for the article by form ascospores that are highly heat-resistant, allowing these King, Michener, and Ito (1969) who reported that ascospores of species of the genera to survive heat sterilization and be present in T. aurantiacus were survived at 88C for 60 min. Therefore, we finished acidic beverages (Samson et al., 2004). analyzed the growth, heat resistance and formation temperature The species of Thermoascus can, as the genus name implies, range of ascospores of Thermoascus spp. in order to establish risk grow at high temperatures. The major species of this genus include analysis data for each Thermoascus spp. Furthermore, we demon- Thermoascus crustaceus, Thermoascus thermophilus, Thermoascus strate that the species of Thermoascus and the closely related aurantiacus, and Thermoascus aegyptiacus (Houbraken & Samson, Byssochlamys genera produce patulin. To evaluate the risk of patulin 2011; Ueda & Udagawa, 1983). These fungi have been detected in production, we analyzed the detection of homology of the gene that encodes isoepoxydon dehydrogenase (idh) an important enzyme in patulin production (Dombrink-Kurtzman & Engberg, 2006; * Corresponding author. Tel.: þ81 43 226 2790; fax: þ81 43 226 2486. Paterson, Kozakiewicz, Locke, Brayford, & Jones, 2003; Puel, E-mail address: [email protected] (T. Yaguchi). Galtier, & Oswald, 2010). A detailed risk analysis based on patulin 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.12.021 8 K. Hosoya et al. / Food Control 41 (2014) 7e12 production was made for the species of Thermoascus grown in beverage model. The ascospores were adjusted to 106 spores/ml in culture medium. standard glucose-tartrate solution, and heat-resistance testing of On the other hand, the detection and identification of fungi has each species was performed using the thermal death time (TDT) been based on morphological observation, and there has been great test tube method (Ueda, Kawara, Yaguchi, & Udagawa, 2010). To interest in a rapid and versatile detection method. Due to the lack of assess the survival, germination rate was determined by counting informative genetic data, a rapid detection method for the species the number of germinated ascospores in triplicate (Ueda & Kawara, of Thermoascus had not previously been established. We success- 2010). A survival curve was plotted from the number of surviving fully developed a PCR method to detect and identify Byssochlamys fungi, and the D-value (min) was calculated. and Neosartorya to the genus and species levels (Hosoya et al., 2012; Nakayama et al., 2010; Yaguchi et al., 2012). 2.4. Evaluation of patulin production in culture and by detection of In this study, we first evaluated the risk of food spoilage on the the isoepoxydon dehydrogenase (idh) gene species of Thermoascus and found the necessity to distinguish Thermoascus spp. from other heat-resistant fungi. Next, we Evaluation for the presence or absence of the idh gene in analyzed various genes used in the phylogenetic classification of members of the Thermoascus genus and related species was per- fungi, designed new specific primers targeting gene sequence formed using the following region-specific primers that encode patterns specifictoThermoascus, and established a PCR method to functional domains (Hosoya et al., 2012): idh2444 (50- ATGCA- rapidly detect and distinguish Thermoascus at the genus level. CATGGAAGGCGAGAC-30) and idh2887 (50-CAAVGTGAATTCCGC- CATCAACCAAC-30). PCR was performed with 1 ml of 5 ng/mlDNA, 2. Materials and methods 1 ml of each primer (10 pmol), 22 ml of DW, and 25 ml of Sap- phireAmp Fast PCR Master Mix (Takara Bio Inc.) subjected to 35 2.1. Phylogenetic analysis of Thermoascus and related species cycles of denaturation at 98 C for 5 s, annealing at 59 C for 5 s, and extension at 72 C for 10 s. Fungi were cultured on potato dextrose agar (PDA) medium The PCR products were electrophoresed on a 2% agarose gel (Eiken Chemical Co., Tokyo, Japan) in the dark at 25 or 37 Cfor7 for 45 min at 100 V, and the presence or absence of bands and days. Fungal DNA was extracted using Dr. GenTLEÔ High Re- band sizes were evaluated. The PCR products were purified using covery Kit (Takara Bio, Inc. Ohtsu, Japan) and adjusted to 5 ng/ml High Pure PCR Product Purification Kits (Roche, Mannheim, in TE buffer. The region coding the large subunit of the RNA Germany). The purified PCR products were labeled with idh2444 polymerase II gene (RPB1) was amplified and the PCR products or idh2887 primers using a Terminator Cycle Sequencing Ready were labeled using the Terminator Cycle Sequencing Ready Re- Reaction Kit (Applied Biosystems). The base sequence was action Kit (Applied Biosystems, Foster City, CA), following the determined using an ABI PRISM 3130 (Applied Biosystems). Ho- method of Samson et al. (2011). The DNA sequences were mology between the idh gene of members of the Thermoascus determined using an ABI PRISM 3130 Genetic Analyzer (Applied genus and Byssochlamys nivea (which produces patulin) was Biosystems). Using the sequences determined here and se- calculated using DNASIS Pro (Hitachi), and patulin production quences from other species of the genus Thermoascus and related potential was evaluated. species obtained through an ARSA search of the DNA Data Bank Patulin production was evaluated in the culture media of Ther- of Japan (DDBJ) (http://arsa.ddbj.nig.ac.jp/html/) and our previ- moascus spp. cultures. The fungi tested were T. thermophilus IFM ously determined sequences, a sequence alignment and neighbor 60075, T. crustaceus IFM 60077, T. aurantiacus IFM 57325, joining trees were prepared using ClustalX software (http:// Byssochlamys verrucosa IFM 48423, and B. nivea NBRC 57325. Fungi clustalx.ddbj.nig.ac.jp/top-.html). was inoculated on 100 ml of Czapek-glucose medium (Wako Pure Chemical Industries, Ltd., Osaka, Japan) or potato dextrose broth 2.2. Growth and ascospore formation of Thermoascus spp. (PDB) (Difco) and cultured in the dark at 35 C for 7 days without agitation. Then, the culture broth containing each fungus was To evaluate the potential for food spoilage due to individual adjusted to pH 3.6 with aqueous acetic acid, extracted with ethyl Thermoascus spp. over a range of temperatures, the growth and acetate, and washed with alkali. Patulin was quantified by high- ascospore formation were examined for the following test fungi: performance liquid chromatography (Nanospace SI-2, Shiseido, T. aegyptiacus IFM 61569, T. aurantiacus IFM 57325, T. crustaceus IFM Tokyo, Japan) fitted with a Mightysil RP-18GP column (Kanto 57326, and T. thermophilus IFM 59664. Each species was inoculated Kagaku and with detection at 290 nm). The detection limit for on oatmeal agar medium (Difco, BD, Sparks, MD) or PDA medium patulin was 0.05 ppm. (Eiken Chemical Co., Tokyo, Japan) in triplicate and cultured at a temperature range of 20e60 C for 14 days.