## 2 Materials and Methods
### Methods
#### 2.2.6 Untargeted Metabolomics Analysis  
Metabolite extraction: An appropriate amount of sample was accurately weighed and placed into a 2 mL centrifuge tube. Subsequently, 600 \(\mathrm{\SIUnitSymbolMicro L}\) of methanol containing 2-chloro-L-phenylalanine (4 ppm) was added, and the mixture was vortexed for 30 s. Then, steel beads were added, and the sample was ground at 55 Hz for 60 s in a tissue grinder, followed by ultrasonication at room temperature for 15 min. After that, it was centrifuged at 12,000 rpm at 4 \({}^{\circ}\)C for 10 min. The supernatant was collected and filtered through a 0.22 \(\mathrm{\SIUnitSymbolMicro m}\) membrane, and the filtrate was transferred into a detection vial for LC-MS analysis. The data were  
\begin{table}
\begin{tabular}{c c c} \hline
**Steps** & **Temperature** & **Digestion Time** \\ \hline
1 & 120 \({}^{\circ}\)C & 3 min \\
2 & 160 \({}^{\circ}\)C & 3 min \\
3 & 190 \({}^{\circ}\)C & 20 min \\ \hline \end{tabular}
\end{table}
Table 1: Sample digestion reference conditions.  
acquired in the MS/MS mode using the data-dependent acquisition (DDA) method. For the research and analysis of mass spectrometry/mass spectrometry (MS/MS) spectra, accurate mass, and isotope patterns, in silico fragmentation was employed. The mass spectrometry analysis was performed by comparing with standard spectral libraries such as the NIST mass spectral library.  
Chromatographic conditions: An ultra-high liquid chromatography system (manufactured by Thermo Fisher Scientific, USA) was utilized. The ACQUITY UPLC\({}^{\circledR}\) HSS T3 column (2.1 \(\times\) 100 mm, 1.8 \(\upmu\)m, produced by Waters, Milford, MA, USA) was employed, operating at a flow rate of 0.3 mL/min, a column temperature of 40 \({}^{\circ}\)C, and an injection volume of 2 \(\upmu\)L. In the positive ion mode, the mobile phase comprised 0.1% formic acid in acetonitrile (B\({}_{2}\)) and 0.1% formic acid in water (A\({}_{2}\)). A gradient elution program was applied: 8% B\({}_{2}\) from 0 to 1 min, 8-98% B\({}_{2}\) from 1 to 8 min, 98% B\({}_{2}\) from 8 to 10 min, 98-8% B\({}_{2}\) from 10 to 10.1 min, and 8% B\({}_{2}\) from 10.1 to 12 min. In the negative ion mode, the mobile phase consisted of acetonitrile (B\({}_{3}\)) and 5 mM ammonium formate in water (A\({}_{3}\)). The gradient elution program was as follows: 8% B\({}_{3}\) from 0 to 1 min, 8-98% B\({}_{3}\) from 1 to 8 min, 98% B\({}_{3}\) from 8 to 10 min, 98-8% B\({}_{3}\) from 10 to 10.1 min, and 8% B\({}_{3}\) from 10.1 to 12 min [20].  
Mass spectrometry conditions: The Thermo Q Exactive mass spectrometer (manufactured by Thermo Fisher Scientific, USA) equipped with an electrospray ionization (ESI) source was used to collect data in both positive and negative ion modes. The spray voltages were set at 3.50 kV and \(-\)2.50 kV, respectively. The sheath gas flow rate was 40 arb, the auxiliary gas flow rate was 10 arb, and the capillary temperature was 325 \({}^{\circ}\)C. The first-level full scan resolution was 70,000 over \(m/z\) 100-1000. The tandem was carried out using HCD at 30 eV with a resolution of 17,500. Unnecessary MS/MS information was dynamically excluded, while the top 10 ions were selected for fragmentation [21]. The isolation window is 2.0 \(m/z\), and the collision energy is 30 eV. These parameters are fixed.