Animal Science Publications Animal Science 2-2018 Autochthonous facility-specific microbiota dominates washed- rind Austrian hard cheese surfaces and its production environment Narciso M. Quijada University of Veterinary Medicine Vienna Evelyne Mann University of Veterinary Medicine Vienna Martin Wagner University of Veterinary Medicine Vienna David Rodríguez-Lázaro Universidad de Burgos Marta Hernández Instituto Tecnológico Agrario de Castilla y León See next page for additional authors Follow this and additional works at: https://lib.dr.iastate.edu/ans_pubs Part of the Bacteriology Commons, Environmental Microbiology and Microbial Ecology Commons, Food Processing Commons, and the Genetics and Genomics Commons The complete bibliographic information for this item can be found at https://lib.dr.iastate.edu/ ans_pubs/525. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html. This Article is brought to you for free and open access by the Animal Science at Iowa State University Digital Repository. It has been accepted for inclusion in Animal Science Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Autochthonous facility-specific microbiota dominates washed-rind Austrian hard cheese surfaces and its production environment Abstract Cheese ripening involves the succession of complex microbial communities that are responsible for the organoleptic properties of the final products. The food processing environment can act as a source of natural microbial inoculation, especially in traditionally manufactured products. Austrian Vorarlberger Bergkäse (VB) is an artisanal washed-rind hard cheese produced in the western part of Austria without the addition of external ripening cultures. Here, the composition of the bacterial communities present on VB rinds and on different processing surfaces from two ripening cellars was assessed by near full length 16S rRNA gene amplification, cloning and sequencing. Non-inoculated aerobic bacteria dominated all surfaces in this study. VB production conditions (long ripening time, high salt concentration and low temperatures) favor the growth of psychro- and halotolerant bacteria. Several bacterial groups, such as coryneforms, Staphylococcus equorum and Halomonas dominated VB and were also found on most environmental surfaces. Analysis of OTUs shared between different surfaces suggests that VB rind bacteria are inoculated naturally during the ripening from the processing environment and that cheese surfaces exert selective pressure on these communities, as only those bacteria better adapted flourished on VB rinds. This study analyzed VB processing environment microbiota and its relationship with VB rinds for the first time, elucidating that the processing environment and the cheese microbiota should be considered as microbiologically linked ecosystems with the goal of better defining the events that take place during cheese maturation. Keywords Cheese rind, bacteria, 16S rRNA gene cloning, Vorarlberger Bergkäse Disciplines Bacteriology | Environmental Microbiology and Microbial Ecology | Food Processing | Genetics and Genomics Comments This is a manuscript of an article published as Quijada, Narciso M., Evelyne Mann, Martin Wagner, David Rodríguez-Lázaro, Marta Hernández, and Stephan Schmitz-Esser. "Autochthonous facility-specific microbiota dominates washed-rind Austrian hard cheese surfaces and its production environment." International journal of food microbiology 267 (2018): 54-61. Posted with permission. Creative Commons License This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License. Authors Narciso M. Quijada, Evelyne Mann, Martin Wagner, David Rodríguez-Lázaro, Marta Hernández, and Stephan Schmitz-Esser This article is available at Iowa State University Digital Repository: https://lib.dr.iastate.edu/ans_pubs/525 *Manuscript with Line Numbers Click here to view linked References 1 Autochthonous facility-specific microbiota dominates washed-rind Austrian hard 2 cheese surfaces and its production environment 3 4 5 Narciso M. Quijada1,2, Evelyne Mann1, Martin Wagner1, David Rodríguez-Lázaro3, 6 Marta Hernández2, Stephan Schmitz-Esser1,4* 7 8 1 Institute for Milk Hygiene, University of Veterinary Medicine Vienna, Vienna, Austria 9 2 Laboratory of Molecular Biology and Microbiology, Instituto Tecnológico Agrario de 10 Castilla y León, Valladolid, Spain 11 3 Division of Microbiology, Department of Biotechnology and Food Science, 12 Universidad de Burgos, Burgos, Spain 13 4 Current address: Department of Animal Science, Iowa State University, Ames, IA, USA 14 15 Keywords: Cheese rind, bacteria, 16S rRNA gene cloning, Vorarlberger Bergkäse 16 17 Corresponding author: 18 Stephan Schmitz-Esser, Department of Animal Science, Iowa State University, 3222 19 NSRIC, 1029 North University Boulevard, Ames, IA, 50011, USA, +1-515-294-2739, 20 [email protected] 1 21 Abstract 22 Cheese ripening involves the succession of complex microbial communities that are 23 responsible for the organoleptic properties of the final products. The food processing 24 environment can act as a source of natural microbial inoculation, especially in 25 traditionally manufactured products. Austrian Vorarlberger Bergkäse (VB) is an 26 artisanal washed-rind hard cheese produced in the western part of Austria without the 27 addition of external ripening cultures. Here, the composition of the bacterial 28 communities present on VB rinds and on different processing surfaces from two 29 ripening cellars was assessed by near full length 16S rRNA gene amplification, cloning 30 and sequencing. Non-inoculated aerobic bacteria dominated all surfaces in this study. 31 VB production conditions (long ripening time, high salt concentration and low 32 temperatures) favor the growth of psychro- and halotolerant bacteria. Several 33 bacterial groups, such as coryneforms, Staphylococcus equorum and Halomonas 34 dominated VB and were also found on most environmental surfaces. Analysis of OTUs 35 shared between different surfaces suggests that VB rind bacteria are inoculated 36 naturally during the ripening from the processing environment and that cheese 37 surfaces exert selective pressure on these communities, as only those bacteria better 38 adapted flourished on VB rinds. This study analyzed VB processing environment 39 microbiota and its relationship with VB rinds for the first time, elucidating that the 40 processing environment and the cheese microbiota should be considered as 41 microbiologically linked ecosystems with the goal of better defining the events that 42 take place during cheese maturation. 43 44 2 45 1. Introduction 46 47 Cheese is one of the oldest fermented foods. There is proven evidence for cheese 48 making in the sixth millennium B.C. and its manufacture is strongly related with 49 regionally different technical, social and economic conditions (Irlinger et al., 2015; 50 Salque et al., 2013;). Particularly in long-ripened raw milk cheeses, cheese 51 fermentation involves a complex and dynamic microbiome that establishes through 52 the ripening process and strongly influences the characteristics of the final products 53 including also food safety, as cheese microbiota can act as a natural barrier for 54 pathogens and spoilage microorganisms (reviewed by Boldyreva et al., 2016). 55 Cheese rinds microbiota differs largely from core cheese microbiota and can develop 56 either by direct inoculation of external ripening cultures and/or by natural colonization 57 of microbes from the processing environment, which is in continuous contact with 58 cheese rinds (Bokulich and Mills, 2012; Irlinger et al., 2015; Monnet et al., 2015). The 59 processing environment encompasses many surfaces such as equipment, brine tanks, 60 ripening rooms, vats, benches, clothes and human skin that can act as potential 61 sources of microorganisms (Kousta et al., 2009; Montel et al., 2014). The successful 62 colonization of these autochthonous microbial communities, summed up in the term 63 “house microbiota”, relies on their ability to cope with environmental conditions such 64 as low temperature, high salt content, humidity, pH, moisture control (brining, 65 pressing), cleaning procedures and competition against other microbes (Bokulich and 66 Mills, 2013; Mounier et al., 2006). Deacidification of the cheese surface by yeasts and 67 molds which consume lactate and produce ammonia as well as extensive washing of 68 cheeses with salt favors the establishment of acid-sensitive and salt- and psychro- 3 69 tolerant bacteria, such as coagulase-negative cocci (CNC, mainly Staphylococcus 70 equorum), Gram-positive coryneforms (Brevibacterium, Corynebacterium) and Gram- 71 negative bacteria such as Halomonas (Coton et al., 2012; Delbes et al., 2007; Gori et 72 al., 2013; Irlinger and Mounier, 2009; Mounier et al., 2005; Rea et al., 2007). Microbial 73 populations involved in cheese ripening are often also found in the processing 74 environment and it has been demonstrated that facility-specific “house” microbiota is 75 implicated in the development of brand-specific characteristics of many traditionally 76 manufactured cheeses (Bokulich and Mills, 2013; Dolci et al., 2009; Mounier et al., 77 2006; Van Hoorde et al., 2010). Moreover, exploring the relationships between 78 ripening and production facility environment can be very useful to understand the 79 impact of the house microbiota