## 3 Results and Discussion
### Analysis and Results
#### 3.1.4 KEGG Enrichment Analysis and Main Differential Metabolites  
The pathways of the differential metabolites between the N1F and TS stages in the Shanghai Lianzhong secondary fermentation method were mainly concentrated in carbohydrate metabolism, amino acid metabolism, membrane transport and other KEGG pathways (Figure 7a1). The enrichment analysis showed that the significantly enriched pathways were synthesis and degradation of ketone bodies (\(p\) < 0.01), arginine biosynthesis (\(p\) < 0.05), pantotheate and CoA biosynthesis (\(p\) < 0.05), alanine, aspartate, and glutamate metabolism (\(p\) < 0.05), beta-alanine metabolism (\(p\) < 0.05), phenylalanine, tyrosine and tryptophan biosynthesis (\(p\) < 0.05), and ABC transporters (Figure 7b1). Figure 5: Score plots and permutation testing under the PLS-DA model for compost metabolites of different groups. (**a1**,**b1**) represent the PLS-DA analysis of N1F-TS comparison groups. (**a2**,**b2**) represent PLS-DA analysis of Phase I-Phase II comparison groups. (**a3**,**b3**) represent PLS-DA analysis of Phase II–Phase III comparison groups. (**a4**,**b4**) represent PLS-DA analysis of the TS-Phase II comparison groups. (**a5**,**b5**) represent PLS-DA analysis of the TS-Phase III comparison groups. PLS-DA model verification: The X-axis represents the permutation retention of the permutation test (Keep the scale consistent with the original model Y variable order. The point with the permutation retention of 1 is the R2 and Q2 values of the original model). The Y-axis represent the values of the permutation test for R2 (green dots) and Q2 (blue triangles). The two dashed lines represent the regression lines of R2 and Q2, respectively. In the tertiary fermentation process of Jiangsu Yuguan, the pathways of differential metabolites between phase I and phase II were mainly concentrated in carbohydrate metabolism and lipid metabolism (Figure 7a2). The enrichment analysis showed that the significantly enriched pathways were synthesis and degradation of ketone bodies (\(p<0.01\)) and butanoate metabolism (\(p<0.05\)) (Figure 7b2). The main differential metabolite involved in the pathway was (R)-3-hydroxybutyric acid, which was significantly decreased from phase I to phase II (\(p<0.01\)) (Table 2). Figure 6: VENN diagrams of differential metabolites among different metabolic groups. (**a**): Venn_ TS vs N1F, Phase I vs Phase II. (**b**) Venn-TS vs N1F, Phase II vs Phase III. (**c**): Venn-TS vs phase III, Phase II vs Phase III. The main differential metabolites involved in the pathway were (R)-3-hydroxybutyric acid, indole, L-aspartic acid, and maltose. (R)-3-Hydroxybutyric acid (\(p<0.01\)), L-aspartic acid (\(p<0.01\)), and indole (\(p<0.01\)) were significantly higher at the TS stage than at the N1F stage for the secondary fermentation method in Shanghai lianzhong, while maltose (\(p<0.01\)) was significantly lower (Table 2). In the tertiary fermentation method of Jiangsu Yuguan, the pathways of the differential metabolites between phase II and phase III were mainly concentrated in membrane transport, amino acid metabolism, carbohydrate metabolism, lipid metabolism, metabolism of cofactors and vitamins and other KEGG pathways(Figure 7a3). Enrichment analysis showed that the significantly enriched pathways were synthesis and degradation of ketone bodies (\(p<0.01\)), vitamin B6 metabolism (\(p<0.05\)), and ABC transporters (\(p<0.05\)) (Figure 7b3). (R)-3-Hydroxybutyric acid and 5-aminolevulin acid had significantly higher contents (\(p<0.01\)), while there were significantly lower contents in pyridoxal (\(p<0.01\)) and maltose (\(p<0.05\)) at phase III than at phase II (Table 2). The pathways including the differential metabolites between the TS stage of the Shanghai Lianzhong secondary fermentation method and phase II of the Jiangsu Yuguan tertiary fermentation method were mainly concentrated in metabolism of cofactors and vitamins, amino acid metabolism, carbohydrate metabolism, membrane transport and other KEGG pathways (Figure 7a4).