Tyrosine

To investigate eumelanin synthesis in F. kingsejongi, cells were grown at 25 °C for 16 days on LB agar plates supplemented with: (1) 1 g/L tyrosine (Sigma-Aldrich, St. Louis, MO, USA), (2) 100 mg/L kojic acid (Sigma-Aldrich), or (3) 1 g/L tyrosine + 100 mg/L kojic acid. As positive and negative controls, eumelanin-producing S. avermitilis 31267^T and non-melanin-producing E. coli TOP10 were grown on LB plates identical to those used to grow F. kingsejongi (25 °C for 7 days for S. avermitilis, 37 °C for 6 days for E. coli). Similarly, pyomelanin synthesis was investigated by growing F. kingsejongi, recombinant E. coli expressing HPPD, and E. coli on LB agar plates supplemented with: (1) 1 g/L tyrosine, (2) 1 g/L tyrosine + 50 μg/L sulcotrione (Sigma-Aldrich, St. Louis, MO, USA), or (3) 1 g/L tyrosine + 100 μg/L sulcotrione.

Standards of phenylalanine (Phe), para-tyrosine (p-Tyr), ortho-tyrosine (o-Tyr), meta-tyrosine (m-Tyr), 3-nitrotyrosine (3-NO2-Tyr), 3-chlorotyrosine (3-Cl-Tyr), 3-iodotyrosine (3-I-Tyr), 8-oxo-2-deoxyguanosine (8-OHdG), 2-deoxyguanosine (2-dG) (96% w/w purity) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Deuterated phenylalanine (Phe-D₅) with a 98% atom D enrichment was purchased from CDN Isotopes (Pointe-Claire, QC, Canada), and it was used as an internal standard. Water was Milli-Q grade (18.2 MV) from a Millipore purification system. Acetonitrile (ACN) (LC-MS grade), methanol (MeOH) (LC-MS grade), and formic acid (analytical grade) were purchased from Sigma Aldrich Química SA (Madrid, Spain).
Creatinine was quantified in urine samples with the enzyme immunoassay (EIA) MicroVue Creatinine kit purchased from Quidel Corporation (Athens, GA, USA) and the spectrophotometer Halo Led 96 from Dynamica Scientific Ltd (Livingston, United Kingdom).

The strains E. faecalis EF37 and ATCC 29212, E. faecium FC12, and FC643 were used in this work. E. faecalis EF37 and E. faecium FC12 were previously isolated from traditional Italian cheese while E. faecium FC643 were isolated from silage. The strains were stored in 20% (w/v) glycerol at -80°C and pre-cultivated for 24 h at 37°C in BHI Broth (Oxoid, Basingstoke, UK) added with 800 mg/l of tyrosine (Sigma–Aldrich, Gallarate, Italy).
After 24 h of pre-cultivation, the microorganisms were inoculated, at a concentration of approximately 6.5 log CFU/ml, in BHI Broth and in Bover-Cid and Holzapfel broth, a medium proposed to highlight the BA formation (BAM; Bover-Cid and Holzapfel, 1999 (link)), added or not with 800 mg/l of tyrosine and incubated at 37°C for 96 h. At defined times (1, 2, 3, 4, 5, 6, 7, 8, 24, 48, 72, and 96 h), the changes of optical density at 600 nm (OD₆₀₀) were monitored. The modification of pH was determined by a pHmeter Basic 20 (Crison Instruments, Barcelona, Spain).
The maximum cell concentration reached in stationary phase was determined after 24 h of incubation by plate counting enterococci onto BHI agar. In addition, 2 ml aliquots of each culture was centrifuged at 3000 rpm for 10 min and the obtained cell pellets were frozen at -80°C.