BRAIN HEART INFUSION BROTH

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Supplement the medium with 800 mg/l of tyrosine -Tubes of Oxoid Brain Heart Infusion Broth which are not used the same day as sterilized should be placed in a boiling water bath for several minutes to remove absorbed oxygen, and cooled rapidly without shaking, just before use.

Protocol tips
-Supplement the medium with 800 mg/l of tyrosine -Tubes of Oxoid Brain Heart Infusion Broth which are not used the same day as sterilized should be placed in a boiling water bath for several minutes to remove absorbed oxygen, and cooled rapidly without shaking, just before use.

Publication protocol

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), 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.

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