## 3 Results  
### Essential Amino Acid and Proteinogenic Amino Acid Profile of the Mushroom Samples  
The total essential amino acid and proteinogenic amino acid contents of the pretreated mushroom samples are presented in Figure 3. The oyster mushroom samples contained 17 protein-building amino acids, with their total amounts ranging from 144.34 (fresh oyster mushroom) to 248.91 mg/g for the pretreated mushroom samples (Appendix A). The essential amino acid content (including arginine) constituted 35-44% of the total amino acids in the mushroom samples, indicating excellent protein quality. All pretreatment methods caused a significant increase in the essential amino acid and total amino acid content of the mushroom samples (\(p<0.05\)). Among these, the microwave pretreatment increased the amino acid concentration the most (72.4%), reaching 248.91 mg/g. Similarly, \begin{table}
\begin{tabular}{l c c} \hline \hline  & **Proteinogenic and Free Amino Acids** & **Biogenic Amines** \\ \hline
**Cation exchange column type** & IONEX OSTION LCP5020 & OSTION LG ANB \\ \hline
**Column size** & 200 mm \(\times\) 3.7 mm & 60 mm \(\times\) 3.7 mm \\ \hline
**Column temperature** & 55 \({}^{\circ}\)C and 65 \({}^{\circ}\)C & 55 \({}^{\circ}\)C and 65 \({}^{\circ}\)C \\ \hline
**Reactor temperature** & 121 \({}^{\circ}\)C & 121 \({}^{\circ}\)C \\ \hline
**Eluent flow rate** & 0.30 cm\({}^{3}\)/min & 0.30 cm\({}^{3}\)/min \\ \hline
**Ninhydrin flow rate** & 0.25 cm\({}^{3}\)/min & 0.25 cm\({}^{3}\)/min \\ \hline
**Sample volume injected** & 100 \(\upmu\)L & 100 \(\upmu\)L \\ \hline
**Detection wavelength** & 440 nm (proline) and 570 nm & 570 nm \\ \hline
**Buffers** & Li-citrate buffers & Na/K-citrate buffers \\ \hline
**Analysis time** & 200 min & 97 min \\ \hline
**Detection limit** & 0.5 \(\upmu\)mol/dm\({}^{3}\) & 0.5 \(\upmu\)mol/dm\({}^{3}\) \\ \hline
**Standard deviation** & 2–5\% & 2–5\% \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Measurement parameters of the automatic amino acid analyzer. the essential amino acid content showed the highest increase in the microwaved samples, rising from 58.05 mg/g to 102.22 mg/g. The total essential amino acid and proteinogenic amino acid contents of the pretreated fermented mushroom samples are presented in Figure 4. In general, fermentation reduced the total amino acid content of the pretreated samples, except in the case of the fresh fermented ones. The total amount of protein-building amino acids in the pretreated fermented samples ranged from 130.86 to 186.96 mg/g (Appendix B). Among these, the fresh fermented oyster mushrooms had the highest total amino acid content at 186.96 mg/g, followed by the blanched fermented samples at 184.36 mg/g, while the steamed fermented samples showed the lowest at 130.86 mg/g. Fermentation affected the essential amino acid content of the mushroom samples differently, causing an increase for the fresh, blanched, and oven-pretreated samples and decrease for others. Among these, the blanched fermented samples showed the highest essential amino acid ratio (44.92%) reaching 82.81 mg/g. Notably, among all the samples, only the steamed fermented ones exhibited a lower essential amino acid content compared to both their pretreated counterparts and the fresh oyster mushrooms. The essential amino acid content (including arginine) constituted 38-45% of the total amino acids in the pretreated fermented mushroom samples. The amino acid profile of the mushroom samples was evaluated using a principal component analysis, as shown in Figures 5 and 6. The first two principal components accounted for 54.12% of the variance, while the first three principal components accounted for 65.80% of the total variance, representing the majority of the dataset's variation. Figure 3: Total essential amino acid (EAA, grey column) and total proteinogenic amino acid (AA, black column) content of the oyster mushroom samples after pretreatments (mean \(\pm\) SE, n = 4). Values with different letters (a–g) differ significantly among total EAA values.