Characterisation of Fungal Contamination Sources for Use in Quality Management of Cheese Production Farms in Korea
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Open Access Asian-Australas J Anim Sci Vol. 33, No. 6:1002-1011 June 2020 https://doi.org/10.5713/ajas.19.0553 pISSN 1011-2367 eISSN 1976-5517 Characterisation of fungal contamination sources for use in quality management of cheese production farms in Korea Sujatha Kandasamy1,a, Won Seo Park1,a, Jayeon Yoo1, Jeonghee Yun1, Han Byul Kang1, Kuk-Hwan Seol1, Mi-Hwa Oh1, and Jun Sang Ham1,* * Corresponding Author: Jun Sang Ham Objective: This study was conducted to determine the composition and diversity of the fungal Tel: +82-63-238-7366, Fax: +82-63-238-7397, E-mail: [email protected] flora at various control points in cheese ripening rooms of 10 dairy farms from six different provinces in the Republic of Korea. 1 Animal Products Research and Development Methods: Floor, wall, cheese board, room air, cheese rind and core were sampled from cheese Division, National Institute of Animal Science, Rural Development Administration, Wanju 55365, Korea ripening rooms of ten different dairy farms. The molds were enumerated using YM petrifilm, while isolation was done on yeast extract glucose chloramphenicol agar plates. Morphologically a Both the authors contributed equally to this work. distinct isolates were identified using sequencing of internal transcribed spacer region. ORCID Results: The fungal counts in 8 out of 10 dairy farms were out of acceptable range, as per Sujatha Kandasamy hazard analysis critical control point regulation. A total of 986 fungal isolates identified https://orcid.org/0000-0003-1460-449X and assigned to the phyla Ascomycota (14 genera) and Basidiomycota (3 genera). Of these Won Seo Park https://orcid.org/0000-0003-2229-3169 Penicillium, Aspergillus, and Cladosporium were the most diverse and predominant. The Jayeon Yoo cheese ripening rooms was overrepresented in 9 farms by Penicillium (76%), while Aspergillus https://orcid.org/0000-0003-3593-5191 in a single farm. Among 39 species, the prominent members were Penicillium commune, Jeonghee Yun https://orcid.org/0000-0001-5929-0055 P. oxalicum, P. echinulatum, and Aspergillus versicolor. Most of the mold species detected Han Byul Kang on surfaces were the same found in the indoor air of cheese ripening rooms. https://orcid.org/0000-0002-8429-2818 Conclusion: The environment of cheese ripening rooms persuades a favourable niche for Kuk-Hwan Seol mold growth. The fungal diversity in the dairy farms were greatly influenced by several factors https://orcid.org/0000-0002-0907-882X Mi-Hwa Oh (exterior atmosphere, working personnel etc.,) and their proportion varied from one to another. https://orcid.org/0000-0001-7838-5260 Proper management of hygienic and production practices and air filtration system would be Jun Sang Ham effective to eradicate contamination in cheese processing industries. https://orcid.org/0000-0003-4966-6631 Submitted Jul 8, 2019; Revised Aug 20, 2019; Keywords: Fungal Diversity; Cheese Ripening Rooms; Dairy Farms; Contaminant Sources Accepted Sept 30, 2019 INTRODUCTION In Korea, both importation as well as domestic consumption of cheese is gradually increas- ing in the consumer market and food processing industry. Domestic cheese manufacturers are more focusing to develop different products leading to an increase in production [1]. Although cheese consumption is safe, still numerous outbreaks of foodborne pathogens are reported from cheese. Risk assessment of cheese spoilage by pathogenic bacteria is well- documented [2]. In contrast, despite data that several molds are involved in the spoilage of dairy products [3,4], few studies have been done on identification and assessment of molds from cheeses and/ their production environment [5-7]. Cheese ripening rooms provide an excellent environment for the growth of several undesirable molds [8]. Penicillium, Aspergillus, Cladosporium, Alternaria, Fusarium, and Talaromyces are most frequently reported genera in cheese spoilage [7,9,10]. In addition, some adventitious molds can also contaminate cheese during the ripening process [11]. These contaminants predominantly originate from the environments including ambient Copyright © 2020 by Asian-Australasian Journal of Animal Sciences This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and repro- 1002 duction in any medium, provided the original work is properly cited. www.ajas.info Kandasamy et al (2020) Asian-Australas J Anim Sci 33:1002-1011 air, equipments, handling persons, raw materials and ingre- Sample preparation and quantitative microbiological dients, and surfaces [12-14]. Contamination of cheese by these analyses molds is unfavourable to cheese quality, affecting the exterior Surface sampling was taken through swabbing an area of 10 appearance, off-flavours and production of toxic secondary cm2 from the floor, wall and cheese board using a pre-wetted metabolites [7,15]. The consumption of cheese contaminated (0.1% w/v peptone water) 3 M Quick swab (3M, St Paul, MN, by molds and or their toxins can pose serious foodborne ill- USA). For ripened cheeses, the rind (9 cm2 on each face) and ness and huge financial losses [16]. In-turn to avoid or delay core (11 g) samples excised aseptically were transferred into mold spoilage and thereby to increase the microbial quality, stomacher bags containing sterile 99 mL physiological saline safety and shelf life of cheese/other dairy products, prevention (pre heated at 45°C) and homogenized (250 rpm, 90 s) using approaches together with hurdle technologies are employed a Stomacher lab blender (FR/Bag Mixer; Interscience, St. Nom, by commercial dairy farms. Normally, most of the traditional France) [19]. Both cheese and surface samples were serially cheeses are manufactured in local dairy farms under poor diluted in peptone water and 1 mL dilution was placed onto hygienic conditions due to lack of awareness of food safety YM petrifilm (3M, USA). The indoor air was evaluated by and systematization [14,17]. Although cleaning and disin- exposing YM petrifilm (3M, USA) for 15 min under normal fection practices considerably lower the microbial load in the air circulation in plants. In all cases, the petrifilms were in- production environment, surfaces are hardly sterile. Occa- cubated under dark conditions at 25°C for 5 days. The total sional examination of the microbiological population or number of viable molds on the surfaces was determined in determination of certain microbial types is required in the colony-forming unit (CFU)/cm2 and propagules on air as dairy industry [12]. The intensity of microbial contamination CFU/plate. along with identification of the most proliferative species is significant, as they might offer a hint of problems within the Isolation of molds from the cheese ripening room production environment besides the potential risk due to the As stated above, the sample preparation and serial dilutions existence of toxin producers [13,18]. In order to guarantee were done and 1 mL dilution of each sample was plated onto a safe food for consumers, the producers have to focus on the the yeast extract glucose chloramphenicol (YEGC) agar me- implementation of suitable production and hygiene practices, dium (g/L yeast extract, 5; glucose, 20; chloramphenicol, 0.1; execution of hazard analysis critical control point (HACCP) and agar, 15) (Sigma-Aldrich, Saint-Louis, MO, USA). The system and to utilize air filtration/decontamination equip- plates were incubated under dark conditions at 25°C for 5 ments. To employ these practices, an essential knowledge of days and based on the morphological difference (shape and the existence and source of the organisms within the dairy colour), individual colonies were selected, restreaked and farm environment (air, equipment and surfaces) is mandatory. further purified on YEGC agar plates. The purified isolates Therefore, we performed an in-depth study on the occurrence grown in YEGC broth were stored at –80°C in 10% glycerol of molds and their diversity associated within the cheese until further use. ripening rooms (wall, floor, cheese board, air and ripened cheese) in the dairy farms of Korea, in line to assess the con- Identification of molds tamination sources and upturn the effectiveness of cleaning/ For internal transcribed spacer (ITS)-polymerase chain reac- disinfection methods and to control the air quality. tion (PCR) based molecular identification, the genomic DNA was extracted from the fungal isolates according to Al-Samar- MATERIALS AND METHODS rai and Schmid [20]. The genomic DNA region covering ITS1- 5.8S-ITS2 was PCR amplified using the universal ITS1 (5′- Selection of Industrial dairy farms and sample collection TCC GTA GGT GAA CCT GCG G - 3′) and ITS4 (5′- TCC Ten dairy farms producing speciality (Gouda and Cheddar) TCC GCT TAT TGA TAT GC -3′) primers [21] and the pu- cheeses were sampled during May 2017 to March 2018 from rified PCR products (~550 bp) were sequenced using Sanger six different provinces of the Republic of Korea. Two farms sequencing method. The obtained sequences were searched were located in Jeonbuk (B, F), three in Jeonnam (A, D, J), for sequence homology against known fungal species at Gen- two in Gyeongnam (E, G) and one from each Chungnam (C) Bank Database using the “basic local alignment tool” (BLAST) Gyeonggi (H), and Gangwon (I). The ripened cheese types after editing and trimming the sequences using Bioedit se- produced from farms A, E, G, H, and J was Gouda, while B, quence alignment editor. C, D, I were Cheddar and Gouda, and F was Cheddar. Sam- ples were collected at various source points (wall, floor, cheese RESULTS AND DISCUSSION board, air and ripened cheese) in the cheese ripening rooms of the dairy farms. All samples were transported to the lab- Enumeration of cultivable molds oratory using a cooler box and analysed within 24 h of arrival. Molds are the most frequent contaminants in cheese indus- www.ajas.info 1003 Kandasamy et al (2020) Asian-Australas J Anim Sci 33:1002-1011 tries, which provide a suitable niche (low pH, high moisture of the cheese environment varies from one farm to another.