## 3 Results and Discussion
### Dynamic Changes in Secondary Metabolites during Fermentation  
The volcano plot (Figure 3A) illustrates the relative abundance differences of metabolites between the CK and JASF groups and the statistical significance of these differences. In this plot, 595 metabolites are significantly upregulated in the experimental group, 497 metabolites are significantly downregulated, and 574 metabolites show no significant difference. The size of the dots indicates the VIP (variable importance in projection) score, with larger dots representing higher VIP scores and suggesting greater importance in distinguishing between the two groups (Table S1). The OPLS-DA S-plot (Figure 3B)provides a visual representation of the metabolites that contribute most significantly to the differences between the CK and JASF groups. Metabolites located closer to the top right and bottom left corners exhibit more significant differences and have VIP values greater than 1, indicating that they are key biomarkers for the fermentation process. These metabolites play a critical role in distinguishing between the CK and JASF groups. Although metabolites with smaller VIP values contribute less to differentiating the two groups, they still affect the overall metabolic profile and may interact with key metabolites. To further expand on the analysis and discussion of Figure 3C, it is crucial to delve deeper into the implications of the observed shifts in secondary metabolites between the CK and JASF groups. The heatmap clearly indicates that the metabolic landscape of Jerusalem artichoke juice undergoes significant changes during fermentation, with distinct metabolites contributing to different stages of the process [55]. The prominence of amino acids and their derivatives in the CK group suggests that these compounds are vital substrates in the early stages, as they may support the growth of specific microbial communities and facilitate initial microbial proliferation. The subsequent decrease in amino acid levels in the JASF group suggests their consumption during fermentation, either for microbial metabolism or for the biosynthesis of secondary metabolites like alkaloids and terpenoids. Organic acids, such as citric, lactic, and succinic acids, are also essential metabolic intermediates, particularly in maintaining pH balance, which directly affects microbial diversity and enzyme activity. The elevated levels of organic acids in the CK group, followed by their reduction in the JASF group, suggest that these acids are metabolized by microorganisms during fermentation, potentially being converted into alcohols and esters that contribute to flavor development. Additionally, phenolic acids, which are abundant in the CK group, play a protective role against oxidative damage and microbial invasion during the initial stages because of their antioxidant properties. However, as fermentation progresses, phenolic acids may be metabolized or transformed into more complex secondary metabolites, such as flavonoids and alkaloids, which exhibit enhanced bioactivity. A notable increase in alkaloids and terpenoids in the JASF group points to a shift in microbial metabolism under more competitive conditions. These metabolites, often associated with stress responses in both plants and microbes, may help shape the microbial community by inhibiting certain microorganisms and promoting the growth of others. Moreover, heterocyclic compounds and flavonoids, which are more abundant in the JASF group, further support the microbial balance and antioxidant mechanisms. Heterocyclic compounds are involved in microbial signaling pathways, while flavonoids, known for their antioxidant activity, may be synthesized in greater amounts during the later stages of fermentation to protect the matrix from oxidative stress and enhance product stability. Figure 3: _Cont._  
The KEGG pathway enrichment analysis highlights significant metabolic pathway changes between the CK and JASF groups during the natural fermentation of Jerusalem artichoke tuber juice (Figure 3D).