Development of a DNA Array for the Simple Identification of Major Filamentous Fungi in the Beverage Manufacturing Environment

Aoyama, Fuyuki; Miyamoto, Takahisa

doi:10.4265/bio.21.161

Cited by 5 publications

(4 citation statements)

References 22 publications

(19 reference statements)

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“…In recent years, DNA microarray‐based methods for the detection and identification of fungi related to the food and beverage industry have been developed and used for multiplex detection (Isshiki et al . ; Aoyama and Miyamoto ). The partial sequences of the isocitrate lyase gene from T. macrosporus and T. trachyspermus (Fig. )…”

Section: Resultsmentioning

confidence: 96%

Design of a species-specific PCR method for the detection of the heat-resistant fungi Talaromyces macrosporus and Talaromyces trachyspermus

Yamashita

Nakagawa²,

Sakaguchi

et al. 2017

Lett Appl Microbiol

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Significance and Impact of the Study: Polymerase chain reaction (PCR)-based detection is rapid, convenient and sensitive compared with traditional methods of detecting heat-resistant fungi. In this study, a PCR-based method was developed for the detection and identification of amplification products from Talaromyces macrosporus and Talaromyces trachyspermus using primer sets that target the isocitrate lyase gene. This method could be used for the on-site detection of T. macrosporus and T. trachyspermus in the near future, and will be helpful in the safety control of raw materials and in food and beverage production. AbstractHeat-resistant fungi occur sporadically and are a continuing problem for the food and beverage industry. The genus Talaromyces, as a typical fungus, is capable of producing the heat-resistant ascospores responsible for the spoilage of processed food products. Isocitrate lyase, a signature enzyme of the glyoxylate cycle, is required for the metabolism of non-fermentable carbon compounds, like acetate and ethanol. Here, species-specific primer sets for detection and identification of DNA derived from Talaromyces macrosporus and Talaromyces trachyspermus were designed based on the nucleotide sequences of their isocitrate lyase genes. Polymerase chain reaction (PCR) using a species-specific primer set amplified products specific to T. macrosporus and T. trachyspermus. Other fungal species, such as Byssochlamys fulva and Hamigera striata, which cause food spoilage, were not detected using the Talaromyces-specific primer sets. The detection limit for each species-specific primer set was determined as being 50 pg of template DNA, without using a nested PCR method. The specificity of each species-specific primer set was maintained in the presence of 1,000-fold amounts of genomic DNA from other fungi. The method also detected fungal DNA extracted from blueberry inoculated with T. macrosporus. This PCR method provides a quick, simple, powerful and reliable way to detect T. macrosporus and T. trachyspermus.

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Section: Resultsmentioning

confidence: 96%

Design of a species-specific PCR method for the detection of the heat-resistant fungi Talaromyces macrosporus and Talaromyces trachyspermus

Yamashita

Nakagawa²,

Sakaguchi

et al. 2017

Lett Appl Microbiol

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“…). A DNA microarray was recently developed for detecting multiple fungi in the same sample (Aoyama and Miyamoto ). Probes were designed based on partial T. macrosporus and T. trachyspermus hydrophobin gene sequences.…”

Section: Resultsmentioning

confidence: 99%

Detection of Talaromyces macrosporus and Talaromyces trachyspermus by a PCR assay targeting the hydrophobin gene

Yamashita

Nakagawa²,

Sakaguchi

et al. 2019

Lett Appl Microbiol

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Talaromyces species are typical fungi capable of producing the heat‐resistant ascospores responsible for the spoilage of processed food products. Hydrophobins, which are unique to fungi, are small secreted proteins that form amphipathic layers on the outer surface of fungal cell walls. In this study, species‐specific primer sets for detecting and identifying Talaromyces macrosporus and Talaromyces trachyspermus were designed based on hydrophobin gene sequences. A conventional polymerase chain reaction (PCR) assay using these primer sets produced species‐specific amplicons for T. macrosporus and T. trachyspermus. The detection limit for each primer set was 100 pg template DNA. This assay also detected fungal DNA extracted from blueberries inoculated with T. macrosporus. Other heat‐resistant fungi, including Byssochlamys, Neosartorya and Talaromyces species, which cause food spoilage, were not detected in PCR amplifications with these primer sets. Furthermore, a conventional PCR assay using a crude DNA extract as the template also yielded amplicons specific to T. macrosporus and T. trachyspermus. The simple and rapid PCR assay described herein is highly species‐specific and can reliably detect T. macrosporus and T. trachyspermus, suggesting it may be relevant for the food and beverage industry. Significance and Impact of the Study The heat‐resistant ascospores of Talaromyces macrosporus and Talaromyces trachyspermus are responsible for food spoilage after pasteurization. Traditional methods for detecting fungal contamination based on morphological characteristics are time‐consuming and exhibit low sensitivity and specificity. In this study, a conventional polymerase chain reaction (PCR) assay based on hydrophobin gene sequences was developed for the specific detection of T. macrosporus and T. trachyspermus. This detection method was simple, rapid and highly specific. These results suggest that the conventional PCR assay developed in this study may be useful for detecting T. macrosporus and T. trachyspermus in raw materials and processed food products.

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“…are heat‐resistant filamentous fungi usually of soil origin and are frequently detected in pasteurized fruit‐based products including flavored mineral waters (Hocking & Pitt, 2001 ; Pitt & Hocking, 2009 ). Species from genus Phoma are common outdoor and soil fungi that have been previously detected also in soft drink manufacturing facilities (Aoyama & Miyamoto, 2016 ).…”

Section: Discussionmentioning

confidence: 99%

Fungal diversity on brewery filling hall surfaces and quality control samples

Sohlberg

Sarlin

Juvonen

2022

Yeast

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Breweries produce an increasing selection of beer and nonbeer beverages. Yeast and filamentous fungi may compromise quality and safety of these products in several ways. Recent studies on fungal communities in breweries are scarce and mostly conducted with culture‐dependent methods. We explored fungal diversity in the production of alcoholic and nonalcoholic beverages in four breweries. Samples were taken for next generation sequencing (NGS) at the key contamination sites in 10 filling lines. Moreover, fungal isolates were identified in 68 quality control samples taken from raw materials, filling line surfaces, air, and products. NGS gave a comprehensive view of fungal diversity on filling line surfaces. The surface‐attached communities mainly contained ascomycetous fungi. Depending on the site, the dominant genera included Candida, Saccharomyces, Torulaspora, Zygosaccharomyces, Alternaria, Didymella, and Exophiala. Sanger sequencing revealed 28 and 27 species of yeast and filamentous fungi, respectively, among 91 isolates. The most common species Saccharomyces cerevisiae, Zygosaccharomyces rouxii, and Wickerhamomuces anomalus were detected throughout production. Filling line surface and air samples showed the greatest diversity of yeast and filamentous fungi, respectively. The isolates of the most common yeast genera Candida, Pichia, Saccharomyces, and Wickerhamomyces showed low spoilage abilities in carbonated, chemically preserved drinks but could grow in products with reduced hurdles. Preservative resistant yeasts were rare, belonging to the species Dekkera bruxellensis, Pichia manschurica, and Zygosaccharomyces bailii. Penicillium spp. were dominant filamentous fungi. The results of this study help to evaluate spoilage risks caused by fungal contaminants detected in breweries.

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Development of a DNA Array for the Simple Identification of Major Filamentous Fungi in the Beverage Manufacturing Environment

Cited by 5 publications

References 22 publications

Design of a species-specific PCR method for the detection of the heat-resistant fungi Talaromyces macrosporus and Talaromyces trachyspermus

Design of a species-specific PCR method for the detection of the heat-resistant fungi Talaromyces macrosporus and Talaromyces trachyspermus

Detection of Talaromyces macrosporus and Talaromyces trachyspermus by a PCR assay targeting the hydrophobin gene

Fungal diversity on brewery filling hall surfaces and quality control samples

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