L histidine monohydrochloride

The mAb (Trastuzumab, Anti HER2 Antibody) was provided by WuXi Biologics. L-histidine monohydrochloride was obtained from Merck. L-histidine and sucrose were gained from Pfanstiehl. Polysorbate 80 (PS80) were purchased from Croda. Type 1 medium borosilicate glass tubing vials (Schott, 2 mL vial, 8 mL vial, 10 mL vial, 20 mL vial, 50 mL vial), stoppers (West, 13 mm, 20 mm) and aluminum covers (West, 13 mm, 20 mm) were used for lyophilization. Syringe of 5 mL and 10 mL were manufactured from Changzhoujinlong Co., Ltd, China.

Table 1 shows the composition of the two synthetic media formulated to determine the ability to form the most toxic BA (HI and TY) and their enhancers (PU and CA): Low Nitrogen Broth (LND), prepared with the objective to decrease the incidence of false positive results of bacteria with a strong peptidase (or deaminase) activity; and the Low Glucose Broth (LGD) developed with the aim to decrease the incidence of false negative responses of bacteria with a great fermentative activity. Before performing the tests both base broth media were supplemented with the precursor amino acids (l-Lysine monohydrate (Merck, Darmstadt, Germany), l-Ornithine monohydrate (Sigma-Aldrich, Steinheim, Germany), l-Histidine monohydrochloride (Merck) and l-Tyrosine disodium salt (Sigma-Aldrich), individually, or adding a mixture of all them (described in the next section as total amino acid broth). The base broth without amino acids added was used as negative control. All media were adjusted to the pH values indicated in Table 1 and autoclaved at 120 °C during 5 min.

To detect bacteria producing biogenic amines, a qualitative test was used [43 (link)]. First, strains were inoculated at 1% (v/v) in modified MRS broth containing 0.1% (w/v) of each amino acid precursor (L-lysine, tyrosine disodium salt, L-histidine monohydrochloride and L-ornithine monohydrochloride (Sigma Aldrich, Darmstadt, Germany)) and incubated at 37 °C for 18 h. The screening was carried out using a specially formulated agar medium (0.5% tryptone, 0.5% yeast extract, 0.5% meat extract, 0.25% NaCl, 0.05% glucose, 0.1% Tween 80, 0.02% MgSO₄, 0.005% MnSO₄, 0.004% FeSO₄, 0.2% ammonium citrate, 0.001% thiamine, 0.2% K₂PO₄, 0.01% CaCO₃, 0.005% pyridoxal-5-phosphate, 1% amino acid precursor, 0.006% bromocresol purple, 2% agar; pH 5.3). For this, 10 µL of each culture was spotted on the medium containing the same amino acids precursor. After 4 days of incubation at 37 °C, a positive result was indicated by the color change and by amino acid precipitation around the corresponding spot for tyramine only. The assays were conducted in duplicate.