## 2 Materials and Methods
### Amino Acid Analysis  
Amino acids were analyzed using reverse-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD). Prior to analysis, the samples were hydrolyzed to amino acids using acid and alkaline hydrolysis [32]. Tryptophan was quantified after alkaline hydrolysis; other amino acids after acidic hydrolysis. For acid hydrolysis, 5 mL of 6 M hydrochloric acid was added to 90 mg of protein sample weighed in a glass tube. The tubes were flushed with nitrogen to exclude oxygen and placed in a 110 \({}^{\circ}\)C oven for 20 h. Cysteine was only partially recovered with this hydrolysis method. Alkaline hydrolysis was conducted similarly, except for the addition of 4.2 M sodium hydroxide instead of hydrochloric acid. All hydrolysates were diluted 1:10 using ultrapure water (1 part hydrolysate + 9 parts water), filtered using 0.2 \(\upmu\)m regenerated cellulose syringe filters (Lab Logistics Group GmbH, Meckenheim, Germany), and stored frozen at \(-80\)\({}^{\circ}\)C until analysis. Acid hydrolyses were performed in duplicate for each of the three process replicates, yielding six replicates in total. Alkaline hydrolyses were performed once for each process replicate. Before derivatization, hydrolyzed samples were further diluted 1:10 in ultrapure water and norvaline and sarcosine were added as internal standards. Amino acids were derivatized according to [PERSON] et al. [33], with the exception of the addition of iodoacetic acid (5 mg/mL in borate buffer) to derivativize cysteine residues [34]. A Shimadzu (Shimadzu Corporation, Kyoto, Japan) LC-20 AD system coupled to a SIL-20 AC autosampler, column oven CTO-20 AC, communication module CBM-20A, and RF-20 Axs fluorescence detector was used for amino acid analysis, along with a Zorbax Eclipse AAA column (150 \(\times\) 4.6 mm, 3.5 \(\upmu\)m; Agilent Technologies, Santa Clara, CA, USA) attached to a precolumn. Chromatographic separation and the detection of amino acids were conducted according to the protocol provided by [PERSON] et al. [33]. Briefly, chromatographic separation was achieved using a gradient of mobile phase A (40 mM NaH\({}_{2}\)PO\({}_{4}\), pH 7.80 \(\pm\) 0.01) and B (45/45/10, _v/v/v_, acetonitrile/methanol/water). Mixtures of commercial amino acid standards were used to prepare an 8-point calibration curve, and amino acids in samples were quantified using calibration curves of external standards and corrected by internal standards. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Fish**} & \multirow{2}{*}{**Process**} & **Protein** & **Protein,** & **Lipids** & **Lipids** \\  & & **(\%, as is)** & **(\%, d.w.)** & **(\%, as is)** & **(\%, d.w.)** \\ \hline \multirow{6}{*}{Roach} & Raw material & \(16.1\pm 0.1\) & 64.9 \(\pm\) 0.3 & 4.2 \(\pm\) 0.0 & 17.0 \(\pm\) 0.1 \\  & Acidic pH shift & 80.1 \(\pm\) 1.0 & 83.0 \(\pm\) 1.6 & 10.7 \(\pm\) 0.5 & 11.1 \(\pm\) 0.4 \\  & Alkaline pH shift & 70.8 \(\pm\) 1.7 & 73.0 \(\pm\) 2.0 & 18.8 \(\pm\) 0.9 & 19.4 \(\pm\) 0.9 \\  & Protamex & 80.4 \(\pm\) 2.2 & 84.0 \(\pm\) 1.5 & 5.5 \(\pm\) 0.1 & 5.8 \(\pm\) 0.1 \\  & Neutrase & 81.3 \(\pm\) 1.6 & 85.1 \(\pm\) 1.5 & 5.7 \(\pm\) 0.2 & 6.0 \(\pm\) 0.2 \\  & Corolase & 79.9 \(\pm\) 1.6 & 83.6 \(\pm\) 1.5 & 6.0 \(\pm\) 0.1 & 6.3 \(\pm\) 0.1 \\ \hline \multirow{6}{*}{B. herring} & Raw material & 15.0 \(\pm\) 0.0 & 63.1 \(\pm\) 0.0 & 7.2 \(\pm\) 0.1 & 29.9 \(\pm\) 0.4 \\  & Acidic pH shift & 79.4 \(\pm\) 0.7 & 80.4 \(\pm\) 0.8 & 16.5 \(\pm\) 1.0 & 16.7 \(\pm\) 1.0 \\ \cline{1-1}  & Alkaline pH shift & 76.0 \(\pm\) 0.9 & 77.9 \(\pm\) 0.7 & 16.4 \(\pm\) 1.8 & 16.8 \(\pm\) 1.8 \\ \cline{1-1}  & Protamex & 87.2 \(\pm\) 1.0 & 91.1 \(\pm\) 1.2 & 3.1 \(\pm\) 0.1 & 3.2 \(\pm\) 0.1 \\ \cline{1-1}  & Neutrase & 84.1 \(\pm\) 1.3 & 87.9 \(\pm\) 0.7 & 3.1 \(\pm\) 0.2 & 3.3 \(\pm\) 0.2 \\ \cline{1-1}  & Corolase & 85.0 \(\pm\) 1.4 & 88.7 \(\pm\) 1.2 & 3.4 \(\pm\) 0.2 & 3.5 \(\pm\) 0.2 \\ \hline \hline \end{tabular}
\end{table}