Process Optimization of Probiotic Fermentation of Birch Sap and Its Metabolome Analysis

Birch sap is a natural plant sap rich in nutrients and bioactive substances, with physiological activities such as antioxidant and anti-inflammatory effects. However, the poor stability of its active components restricts its industrial application. Probiotic fermentation can effectively improve the quality and functional properties of plant-based beverages. Lactobacillus paracasei is widely used in the development of functional foods due to its strong metabolic activity and high safety. Nevertheless, studies on the fermentation process and metabolic mechanism of birch sap using this strain remain insufficient. To enhance the antioxidant activity and processing applicability of birch sap, this study systematically optimized the fermentation process and conducted metabolic mechanism analysis. Using birch sap as the substrate, fermentation was carried out by inoculating Lactobacillus paracasei LC37 to obtain fermented broth. The DPPH free radical scavenging activity was employed as the primary evaluation index. Single-factor experiments combined with response surface methodology (RSM) were applied to optimize the fermentation conditions. Non-targeted metabolomic analysis of samples before and after fermentation was performed using liquid chromatography-mass spectrometry (LC-MS), and orthogonal partial least squares discriminant analysis (OPLS-DA) was used to screen key differential metabolites, followed by KEGG pathway enrichment analysis. The results show that the optimal fermentation conditions are fermentation temperature 37 ℃, fermentation time 41.5 h, initial pH 7, and inoculum size 1.30% (volume fraction). Under these conditions, the DPPH radical scavenging rate reaches 22.91%, which is 3.7 times higher than that of the unfermented sample (4.86%). A total of 505 key differential metabolites were identified before and after fermentation (317 upregulated and 188 downregulated). KEGG analysis indicated significant enrichment of D-fructose-6-phosphate in the pentose phosphate pathway. This enrichment likely contributes to the enhanced antioxidant capacity of the fermented birch sap by supplementing C6 substrates and activating the oxidative phase, thereby promoting the generation of NADPH and ribose-5-phosphate, which provide a metabolic basis for the improved antioxidant activity.