E.Dugat-Bony,ct al. 95 mm Tal:462 nm 100nm Fig.6.Electron microg aphs of rept The viral fraction was obtained with protocol P4. 3.4.Bacteriophage morpholoe ared using the ext er than microbial cells (Aherfi et al 2016).N es are ger 3phage particles( +d4)n al161.3( 7)nm in ng DNA e studies red that add to I loctis phage949 aps d microbial contamin n of the c eviral fractio in the virome profile with en capsid of51 at 10n specific tech skills and la id and 4.Discussion 2) cing,protocos for iral ent per of bacterial cells th ed sample pret vailable for digest ent to d fro finally virus purification(Thurber et al Fach step has step ntion tha viruse n particula is a compl o 009- Compa g the Ep acid ext ols for re or not,indicated a loss of par ticles upon chloroform trea etal., step of the after densit d pr proposed by Ca d th rh by a lin e We decided the potential benefit final DNA yields 3.4. Bacteriophage morphologies Transmission Electronic Microscopy (TEM) analysis was carried out on a sample prepared using the extraction procedure P4 and re￾presentative pictures are presented in Fig. 6. Frequencies of the dif￾ferent morphotypes were calculated based on the analysis of 25 pictures totaling 43 phage particles (Table S6). The most frequent morphotype (79%) exhibited an icosahedral capsid 60.7 ( ± 4.4) nm in diameter, and a noncontractile tail 161.3 ( ± 25.7) nm in length, as illustrated in Fig. 6C, D and E. A larger bacteriophage presenting characteristics si￾milar to Lactococcus lactis phage 949, i.e. an icosahedral capsid of 89 ( ± 6) nm diameter and a noncontractile tail of length 482 ( ± 36) nm, was observed with a frequency of 9% (Fig. 6F). Two other morphotypes were also detected, one with a capsid of 71 ( ± 2) nm diameter and a noncontractile tail of length 173 ( ± 6) nm (Fig. 6B) and the other with a capsid of 51 ( ± 1) nm diameter and a tail of length 140 ( ± 8) nm, at frequencies of 7 and 5%, respectively. 4. Discussion With the increasing attraction for virome sequencing, protocols for extracting viruses from diverse environmental samples have been de￾veloped during the past decade. Prior to nucleic acid extraction and sequencing, these protocols usually included sample pretreatment to make viral particles accessible for extraction, virus concentration and finally virus purification (Thurber et al., 2009). Each step has to be adapted, according to the type of samples studied and the type of virus targeted. For cheese, we chose to blend the samples after a dilution step in trisodium citrate, in order to maximize the chance for recovering viral particles from the matrix. Indeed, citrate is a complexing 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). In order to complete our control condition (protocol P1), we then followed the first steps of the PEG-based protocol proposed by Castro-Mejía et al. (2015), namely centrifugation and PEG precipitation, considering that it were already validated and provided very high recovery rates for all the tested spiked-phages. We decided to evaluate the potential benefit of adding a filtration step (0.22 μm) on the quality of viral fractions and of the resulting virome. Filtration is preferably avoided in virus extraction protocols from environmental sources because some viruses can be very large, even larger than microbial cells (Aherfi et al., 2016). Nevertheless, our choice was motivated by the fact that (i) such large viruses are gen￾erally hosted by organisms which are not part of the cheese ecosystem such as amoebas, protists and microalgae (ii) it ensures complete re￾moval of microbial cells which generally account for the major source of contaminating DNA sequences in virome studies (Roux et al., 2013). Our results demonstrated that adding filtration to the extraction pro￾cedure reduced microbial contamination of the cheese viral fractions by microbial cells, without major modification neither in the particle counts nor in the final virome profile. The best way to purify viruses includes a density gradient ultra￾centrifugation step (Thurber et al., 2009), in which viruses are sepa￾rated from other components of the extract based on their physical properties. This technique is, however, very time-consuming, expensive and requires specific technical skills and lab equipments. Chloroform treatment represents a possible alternative for rapid and efficient virus purification (Biller et al., 2017; Guo et al., 2012) and has already been used in the viral metagenomic context (Willner et al., 2011). This sol￾vent permeabilizes the membranes of bacterial cells and membrane vesicles, disrupting their structural integrity and making nucleic acids available for digestion by nucleases. Our results indicate that such treatment, in combination with filtration, is mandatory for successful quantification of viruses extracted from cheese samples by inter￾ferometry. Even if viral capsids are said to be resistant to chloroform (Biller et al., 2017; Jurczak-Kurek et al., 2016), it is important to mention that some viruses, in particular enveloped viruses, might be lost after such treatment (Biller et al., 2017; Forterre et al., 2013; Thurber et al., 2009; Weynberg et al., 2014). Comparing the Epoisses cheese viral fractions obtained with procedures including chloroform treatment or not, indicated a loss of particles upon chloroform treat￾ment (as illustrated in Fig. 2). Analyzing the nanoparticle counts in the viral band of one Epoisses cheese after density gradient ultra￾centrifugation and dialysis indicated that we recovered approximately ten times less nanoparticles in chloroform-treated samples versus non￾treated samples. However, this was accompanied by a limited reduction of the final DNA yields (< 3 times less DNA in average for treated Fig. 6. Electron micrographs of representative phages from Epoisses cheese. The viral fraction was obtained with protocol P4. E. Dugat-Bony, et al. Food Microbiology 85 (2020) 103278 8