To induce peptidases production at 2, 4, 7, and 10°C, Pseudomonas spp. isolates were first incubated in 5 mL of UHT milk [Modern Farming (Group) Co., Ltd.] for 5 days. Then the proteolytic activity was determined according to the protocol described by Caldera et al. (2016) (link) to quantify the native proteolytic activity at different temperatures.
The trinitrobenzenesulfonic acid (TNBS) method was used to monitor the presence of free α-amino groups, indicators of protein hydrolysis (Polychroniadou, 1988 (link); Marchand et al., 2009a (link)). The TNBS reagent (Sigma–Aldrich, Taufkirchen, Germany) was reacted with the released α-amino groups at pH 9.2 in the dark for 100 min. The intensity of the yellow-orange color of the reaction products was measured by absorption values at 420 nm (VarioskanTM Flash Multimode Reader, Thermo Fisher Scientific, Waltham, MA, United States). Native proteolytic enzyme levels in raw milk were determined from the absorption ratios. Three independent replicate measurements were performed. Pseudomonas spp. isolates were considered peptidase active if the measured absorption exceeded 2 μmol glycine equivalents per mL. Standard curve was generated using glycine (Sigma–Aldrich).
The trinitrobenzenesulfonic acid (TNBS) method was used to monitor the presence of free α-amino groups, indicators of protein hydrolysis (Polychroniadou, 1988 (link); Marchand et al., 2009a (link)). The TNBS reagent (Sigma–Aldrich, Taufkirchen, Germany) was reacted with the released α-amino groups at pH 9.2 in the dark for 100 min. The intensity of the yellow-orange color of the reaction products was measured by absorption values at 420 nm (VarioskanTM Flash Multimode Reader, Thermo Fisher Scientific, Waltham, MA, United States). Native proteolytic enzyme levels in raw milk were determined from the absorption ratios. Three independent replicate measurements were performed. Pseudomonas spp. isolates were considered peptidase active if the measured absorption exceeded 2 μmol glycine equivalents per mL. Standard curve was generated using glycine (Sigma–Aldrich).