2.3.2 Degree of protein hydrolysis
The degree of protein hydrolysis was measured as the released free amino groups using the 2,4,6-trinitrobenzene sulfonic acid (TNBS) method according to Adler-Nissen (1979) and Jung et al. (2005). In brief, a freeze-dried meal containing 1 g of protein (corrected by protein content) was added to distilled water, followed by stirring for 30 min at room temperature (21-23℃). The mixture was centrifuged (Sorvall RC-6 Plus centrifuge, Thermo Scientific, Asheville, NC, USA) at 6,000 ×g for 15 min at room temperature, and the supernatant was collected. A 250-μL aliquot of the supernatant was added to 2 mL 20 mM sodium phosphate buffer (pH 7.8), followed by the addition of 2 mL 0.01% TNBS solution. After mixing using a vortex for 15 s, the mixture was covered by aluminum foil and placed in a water bath at 50℃ for 1 h. The reaction was stopped by adding 4 mL 0.1 N HCl, and the sample was allowed to cool for 10 min. The absorbance of the reaction mixture was measured at 340 nm using a spectrophotometer (Genesys 10S UV-VIS, Thermo Scientific, Madison, WI, USA). The total protein hydrolysis achieved by hydrolyzing proteins with acid was also measured as part of the degree of hydrolysis calculations. The total protein hydrolysis was determined by adding 24 mg of sample (corrected by protein content) to a screw cap Pyrex tube containing 15 mL of 6.0 N HCl. The tubes were incubated at room temperature for 20 h. The dispersion was adjusted to pH 7.0 with 2 M NaOH, followed by filtration through the Whatman Grade 3 filter paper (Whatman International Ltd., Maidstone, UK). A 250 µL aliquot of the above sample was then added to 2.0 mL 1% SDS solution in buffer. This was followed by the addition of 250 µL of the mixed solution to 2.0 mL 10 mM sodium phosphate buffer (pH 7.8). This mixed solution was performed in triplicate and analyzed according to the procedures above. The sample blank was prepared by adjusting a solution of 6.0 N NaOH and 6.0 N HCl to pH 7.0. A 1.5 mM glycine solution was used to create the standard curve to calculate the \(\alpha\)-NH2-glycine equivalents of each sample for use in the following equations to calculate the degree of protein hydrolysis:
\(h=\left(h_{t}-\ h_{c}\right)\ \times\ DF\) (1)
\(DH=\left(\frac{h}{h_{t}}\right)\ \times\ 100\%\) (2)
Where \(h\) is the net mM concentration of\(\alpha\)-NH2-glycine equivalents, \(h_{t}\) is the mM concentration of \(\alpha\)-NH2-glycine equivalents at the time of testing, \(h_{c}\) is the mM concentration of\(\alpha\)-NH2-glycine equivalent at the time of initial inoculation before the microorganism was added, htot is the mM concentration of \(\alpha\)-NH2-glycine equivalents after total acid hydrolysis, and DF is the dilution factor. Measurements were made in triplicate on each of the replicate batches (n=3) and reported as the mean ± one standard deviation.