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.