2.2. Determination of Histamine and Tyramine Degradation by LAB Strains

Degradation of toxic BAs by LAB strains was assessed following the method developed by Leuschner et al. [43], with slight modifications. To prepare resting cells, 10 µL of glycerol stock of each LAB strain were inoculated in 5 mL of MRS broth. After aerobic incubation at 37 °C for 48 h, a loopful of the cultured broth was streaked on MRS agar, which was incubated under the same conditions. A single colony was inoculated in 5 mL of MRS broth. After aerobic incubation at 37 °C for 48 h, 200 µL of the cultured broth was transferred into 10 mL of MRS broth. After aerobic incubation at 37 °C for 48 h under shaking at 200 rpm, the cultured broth was centrifuged at 9000× g for 10 min at 4 °C. The pellet was washed twice with sodium phosphate buffer (0.05 M, pH 7.00; 4.0962 g of Na₂HPO₄ and 2.537 g of NaH₂PO₄ dissolved in 1 L of distilled water, all from Sigma–Aldrich Chemical Co., St. Louis, MO, USA). The cell pellet was resuspended in 10 mL of the same buffer but that contained 0.5 mM histamine and 0.5 mM tyramine (all from Sigma–Aldrich, w/v). The cell suspension was reacted with shaking at 200 rpm for 24 h at 30 °C, an optimal temperature for amine oxidase activity. The cell suspension was then filtered through a 0.2 µm membrane filter (Millipore Co., Bedford, MA, USA) using a syringe and immediately analyzed by HPLC for toxic BA degradation.

To accelerate the screening of toxic BA-degrading LAB strains, an alternative method to measure toxic BA degradation by LAB strains in MRS broth was used [39]. Briefly, after activation of the LAB strains in MRS broth under the same incubation conditions above, 200 µL of the cultured broth were inoculated into 10 mL of MRS broth supplemented with 50 ppm (w/v) of histamine or tyramine. The MRS broth containing histamine or tyramine without bacterial cells was used as a control. All broths were incubated statically at 30 °C for 24 h. Subsequently, the broth culture was filtered through a 0.2 µm membrane and one milliliter of the filtered broth culture was immediately analyzed using HPLC. After assessing the degradation activity of LAB strains using this method, selected LAB strains showing higher degradation activity were retested for degradation activity using the method of measurement in buffer as described above.