5810 benchtop centrifuge

Enterococcus faecalis phage 156 was isolated from 1 g of sample cheese by enrichment culture and following the standard spot method in double-layer agar plates (Ladero et al., 2016 (link)). Enrichment cultures were inoculated with 100 μl of an overnight culture of host bacteria (E. faecalis strains 18a, 19a, 23a, 28a, 63c, BA62, HFS 25, HFS59, CECT 4039, and CECT481^T; Table 1) and incubated without aeration at 37°C for 24 h. Samples were then placed in tubes and centrifuged (2000 ×g for 15 min) in a 5810 Eppendorf benchtop centrifuge, and 100 μl of the supernatant added to a new enrichment culture. After two rounds of enrichment, 10 μl were spotted onto double-layered agar MC-GM17 plates and incubated for 24 h at 37°C. When an inhibition halo was observed, the source supernatant was streaked to obtain single plaques. Some of these plaques were individually tested against the targeted host strains. For bacteriophage purification, a single plaque was picked up with a sterile tip, inoculated into 50 ml of MC-GM17 broth inoculated with the host strain, and incubated at 30°C until cell lysis was observed. The culture was then centrifuged (10,000 ×g for 15 min) in a 7780 centrifuge (Kubota, South Korea) with an AG6512C rotor, concentrated using the PEG/NaCl method (Binetti et al., 2005 (link)), and stored in SM buffer.

Biogenic amines were determined by ultra-high performance liquid chromatography (UHPLC). In broth they were quantified directly from a 100 μl sample, as described by Ladero et al. (2015) (link). The BA in cheese samples were first extracted and quantified as previously described (Herrero-Fresno et al., 2012 (link)) with some modifications. Briefly, 1 g of cheese was mixed with 10 mL of 0.1 M HCl containing 0.2% (w/v) 3,3′thiodipropionic acid using an Ultra Turrax T50 homogeniser (OMNI International, United States) for 2 min at 20,000 rpm. The samples were then disrupted for 30 min in an ultrasonic bath and centrifuged at 5000 ×g for 30 min. After removing the fat layer, the supernatant was filtered through 0.45 μm cellulose acetate filters (VWR, Spain). The filtrates were deproteinised using ultra-filtration inserts (Amicon Ultracel-3K, Millipore) during centrifugation at 3500 g for about 1 h in a 5810 Eppendorf benchtop centrifuge (Eppendorf, Spain). Supernatant samples (100 μL) were then derivatised with diethyl ethoxymethylenemalonate as described by Redruello et al. (2013) (link), and the BA separated out and quantified in an H-Class Acquity UPLC^TM UHPLC system (Waters, United States) running Empower 2 software (Waters), using the conditions described in the latter paper.

Tyramine was determined by UHPLC. In broth it was quantified directly, from a 100 μl sample, as described by Ladero et al. (2015) (link). The tyramine in cheese samples was first extracted and quantified as previously described (Herrero-Fresno et al., 2012 (link)) with some modifications. Briefly, 1 g of cheese was mixed with 10 ml of 0.1 M HCl containing 0.2% (w/v) 3,3′ thiodipropionic acid using an Ultra Turrax T50 homogeniser (OMNI International, USA) for 2 min at 20,000 rpm. The samples were then disrupted for 30 min in an ultrasonic bath and centrifuged at 5000 × g for 30 min. After removing the fat layer, the supernatant was filtered through 0.45 μm PTFE filters (VWR, Spain). The filtrates were deproteinized by precipitation with trichloroacetic acid (12% v/v). They were then kept in ice for 30 min and centrifuged (12,000 × g for 10 min) in a 5810 Eppendorf benchtop centrifuge (Eppendorf, Spain), and the supernatant neutralized with NaOH (0.7 N) as described by del Rio et al. (under review). Supernatant samples (100 μL) were then derivatized with diethyl ethoxymethylenemalonate as described by Linares et al. (2013) (link), and the tyramine separated out and quantified in an H-Class Acquity UPLC^TM UHPLC system (Waters, USA) running Empower 2 software (Waters), as described by Redruello et al. (2013) (link).