E.Dgat Bony,l. most 2.Material and methods 2.1.Sampling procedure et al 012),b such a the pres a (EP,w up a dedicated pro e.ldeally,the method ld b ro duced at the ate ar e produce ere pu hasec mples)to large pro col follow d the PEG-ba d by nik,S e ol by spiking with ated bype ery at the different steps of the pro y no n at Lactic ere enu ple susper re were en erated onye riph mg/l)after 35 days of in ygradients for viral fraction's removal (ille 201. We 2.3.Extraction of the viral fraction from cheese oft出 and ourity. ally,we se itrate(%w/v)into asterile bag and ination in virome se ncing data 2008- tion was ce fuged at300×gfor10mi at 4 to pellet microb e At this Cheese surface(6 g) Camembert(CAM) Epoisses(EP) Saint-Nectaire(SN) 1.Homogenization in Sodium Citrate solution (20 g/I) 2.Centrifugation:300 x e for 5 min:5.000 xg for 45 min were performe 3.Dilution 1:5 in SM Buffer 5.Concentration:PEG Precipitation 4.Filtration:0.45 um+0.22 um 6.Chloroform 5.Concentration:PEG Precipitation P4 Kleiner et al., 2015; Thurber et al., 2009). For food samples, most available procedures have been designed for the recovery of foodborne viruses potentially affecting human health such as noroviruses, rota￾viruses (RoV) and hepatitis viruses (Stals et al., 2012), but not for mi￾crobial viruses such as bacteriophages. Furthermore, because the cheese surface has peculiar characteristics such as the presence of caseins at high concentration, high fat and salt content, it proves necessary to set up a dedicated procedure. Ideally, the method should be easy-to-use and rapid enough to be compatible with medium (dozens to hundred samples) to large-scale studies (hundreds to thousands samples) such as those performed to describe microbial communities (Wolfe et al., 2014). Taking into account all these constraints, we compared four pro￾cedures for the isolation of viruses from cheese surfaces. The backbone of the protocol followed the PEG-based protocol already evaluated by Castro-Mejia et al. on fecal samples (Castro-Mejía et al., 2015), which was also used for dairy bacteriophages recovery from whey after some adaptation (Muhammed et al., 2017). The authors evaluated phage recovery at the different steps of the protocol by spiking with known phages, and concluded that virtually no spiked phages were lost until the ultracentrifugation step (density gradient). In our experimental design, presented in Fig. 1, protocol P1 was constructed from the first steps of this protocol except the replacement of the buffer used for sample suspension, and was thus considered as a control maximizing phage recovery. In parallel, we tested both the effect of adding a fil￾tration step before PEG precipitation in order to deplete microbial cells (protocol P3) and/or the substitution of the expensive and time-con￾suming density gradients for viral fraction's purification by a simple chloroform treatment (protocols P2 and P4) which is used for mem￾brane vesicles removal (Biller et al., 2017; Forterre et al., 2013). We evaluated the usefulness of the four procedures for cheese virome analysis (limited to the viruses with DNA genomes, expectedly the most abundant) using three different types of cheese, namely Camembert, Epoisses and Saint-Nectaire, and following two criteria: particles re￾covery and particles purity. Finally, we selected Epoisses cheese for producing the first cheese surface virome and assessing the level of microbial DNA contamination in virome sequencing data. 2. Material and methods 2.1. Sampling procedure Three types of French surface-ripened cheese were studied, namely Camembert (CAM, bloomy rind), Epoisses (EP, washed rind) and Saint￾Nectaire (SN, natural rind). For each type of cheese, three cheeses produced at the same date and from a unique producer were purchased and analyzed as replicates. Rind was gently separated from the core using sterile knives, and mixed using a blender. 2.2. Microbiological analysis One gram of the cheese surface was diluted 1:10 in sterile saline solution (9 g/l NaCl) and homogenized with an Ultra Turrax Homogenizer (Labortechnik, Staufen, Germany) at full speed for 1 min. Serial dilutions were performed in 9 g/l NaCl and microorganisms were enumerated by surface plating in duplicate on specific agar base medium. Cheese-surface bacteria were enumerated on brain heart in￾fusion agar (Biokar Diagnostics) supplemented with 22.5 mg/l am￾photericin B after 3–5 days of incubation at 25 °C under aerobic con￾ditions. Lactic acid bacteria were enumerated on de Man-Rogosa￾Sharpeagar (pH 6.5, Biokar Diagnostics) supplemented with 22.5 mg/l amphotericin B after 3 days of incubation at 30 °C under anaerobic conditions. Finally, fungal populations were enumerated on yeast ex￾tract-glucose-chloramphenicol (Biokar Diagnostics) supplemented with 2,3,5-triphenyltetrazolium chloride (10 mg/l) after 3–5 days of in￾cubation at 25 °C under aerobic conditions. 2.3. Extraction of the viral fraction from cheese Four protocols, named P1, P2, P3 and P4, were tested in parallel starting from the same material (Fig. 1). Six grams of cheese rind was diluted 1:10 with cold trisodium citrate (2% w/v) into a sterile bag and mixed for 1 min using a BagMixer (Interscience). Citrate is a com￾plexing agent for calcium and allows casein solubilization. It is largely used in nucleic acid extraction protocols for recovering microbial cells from casein network in milk and cheese (Randazzo et al., 2002; Ulve et al., 2008). The solution was centrifuged at 300×g for 10 min at 4 °C in order to pellet big aggregates. The supernatant was centrifuged at 5000×g for 45 min at 4 °C in order to pellet microbial cells. At this Fig. 1. Schematic representation of the experimental proce￾dure for the extraction of viruses from cheese. The surface of three types of cheese was processed according to four routes named P1 to P4. Steps 1 (homogenization), 2 (centrifuga￾tion), 3 (dilution) and 5 (concentration) are common to all protocols, contrary to steps 4 (filtration) and 6 (chloroform treatment) which are optional. For each cheese type, three biological replicates (independent cheeses purchased at the same date and from a unique producer) were performed leading thus to 12 samples per cheese-type. E. Dugat-Bony, et al. Food Microbiology 85 (2020) 103278 2