2.8. Evaluation of Antimicrobial Activity

The antimicrobial activity was determined by measurement of the minimum inhibitory concentration (MIC) according to a previous study [25]. Briefly, the hydrolysates and the peptide fractions were tested against the growths of Gram-positive (Micrococcus luteus ATCC 4698 and Listeria innocua ATCC 33090) and Gram-negative bacteria (Escherichia coli ATCC 25922 and Salmonella enteritidis ATCC 13076), broadly described as inhibited by the bioactive peptides contained in the bHb hydrolysates [2]. A total of 20 µL of strain preculture was inoculated into 10 mL of liquid Lysogeny Broth (LB) medium and the preculture was performed on a rotary shaker (160 min⁻¹) at 37 °C to obtain a standard cell concentration of 10⁶ CFU·mL⁻¹ (colony-forming unit). A total of 100 µL of preculture was distributed in a microtiter plate well and added to 100 µL of the peptide fraction or hydrolysate. Each peptide sample was tested at least in triplicate.

The absorbance of each well was read at 630 nm against the blank obtained before incubation by using a microplate absorbance reader ELx808 with the Gen5 software (Biotek Instrument, Winooski, USA). MIC was the lowest peptide concentration that inhibited the strain growth after incubation (24 h, 37 °C) and was expressed in µg_peptide·mL⁻¹.