## 3 Results and Discussion
### Analysis and Results
#### 3.1.4 KEGG Enrichment Analysis and Main Differential Metabolites
The enrichment analysis showed that the pathways of significant enrichment were synthesis and degradation of ketone bodies (\(p<0.01\)), vitamin B6 metabolism (\(p<0.001\)), lysine biosynthesis (\(p<0.01\)), aminoacyl-tRNA biosynthesis (\(p<0.01\)), arginine biosynthesis (\(p<0.05\)), biotin metabolism (\(p<0.05\)), alanine, aspartate, and glutamate metabolism (\(p<0.05\)), pantothene and CoA biosynthesis (\(p<0.05\)), and ABC transporters (\(p<0.001\)) (Figure 7b4). Compared to the Phase II stage, (R)-3-hydroxybutyric acid, pyridoxal, L-aspartic acid and L-lysine had significantly higher contents (\(p<0.01\)), while pyridosamine 5-phosphate (\(p<0.01\)) and maltose (\(p<0.05\)) had significantly lower contents at the TS stage (Tables 2 and S2). Figure 7: _Cont_. Figure 7: KEGG pathway and enrichment analysis of differential metabolites of the metabolic set. (**a1–a5**) represent the path analysis between comparison groups at different stages, and (**b1–b5**) represent the functional annotations between comparison groups at different stages. \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**Metabolic Set** & **Different Metabolite** & **VIP, PLS-DA** & **FCOY/NO** & \(p\)**-Value** & **KECG Compound** & **KECG Compound** & **KECG Pathway Description** \\ \hline NiF vs. TS & (R)-34 Hydroxybutyrate acid & 2.6707 & 73.8734 & \(6.59\times 10^{-5}\) & Organic acids & Carboxyl acids & Synthesis and degradation of ketone bodies; \\  & Indide & 2.9484 & 1.8832 & \(2.78\times 10^{-4}\) & - & - & \begin{tabular}{c} Transplantitating tyrosine and tryptophan \\ \\ \end{tabular}  \\  & L-Apartic acid & 1.1376 & 17.3029 & \(4.85\times 10^{-5}\) & - & - & \begin{tabular}{c} Lysineosynthesis beta-Alamine metabolism; \\ \end{tabular}  \\  & L-Apartic acid & 1.1376 & 17.3029 & \(4.85\times 10^{-5}\) & \begin{tabular}{c} Symptoms and \\ transmitters \\ \end{tabular}  & \begin{tabular}{c} Amino acids; \\ Instruments \\ \end{tabular}  & \begin{tabular}{c} Amino acids; \\ Instruments \\ \end{tabular}  & \ \begin{tabular}{c} Amino, aspartate and glutamate metabolism; \\ \end{tabular}  \\  & Miltose & 1.0904 & 0.3311 & \(1.27\times 10^{-5}\) & Carboxylids & Oligosaccharides &
\begin{tabular}{c} Hyp  
The pathways of the differential metabolites between the TS stage of the Shanghai Lianzhong secondary fermentation method and the Phase III stage of Jiangsu Yuguang tertiary fermentation method were mainly concentrated in amino acid metabolism, metabolism of cofactors and vitamins, and membrane transport(Figure 7a5). Enrichment analysis showed that the pathways of significant enrichment were synthesis and degradation of ketone bodies (\(p<0.01\)); arginine biosynthesis (\(p<0.05\)); glycine, serine and threonine metabolism (\(p<0.01\)); alanine, aspartate and glutamate metabolism (\(p<0.05\)); vitamin B6 metabolism (\(p<0.05\)); pantothene and CoA biosynthesis (\(p<0.05\)); and ABC transporters (\(p<0.05\)) (Figure 7b5). Compared to phase III, (R)-3-hydroxybutyric acid, L-aspartic acid and pyridoxal had significantly higher contents (\(p<0.01\)), while 5-aminobenzvalinic acid had significantly lower contents at the TS stage (\(p<0.01\)) (Table 2). Some of the main and significantly KEGG enrichment pathways and differential metabolites between the above fermentation stages are shown in Figure 8. For the differential metabolites in synthesis and degradation of ketone bodies, (R)-3-hydroxybutyric acid, (See Figure 8a); for the differential metabolites of glycine, serine and threonine metabolism, L-Aspartate (See Figure 8b); for the differential metabolites of lysine biosynthesis, L-lysine, (See Figure 8c); for the differential metabolites of vitamin B6 metabolism, Pyidoxal, (See Figure 8d), and for the differential metabolite of arginine biosynthesis, Aspartate, (See Figure 8e))... these red-marked differential metabolites were significantly increased between different fermentation stages.