Within the realms of food and traditional Chinese medicine, researchers have directed their efforts toward investigating the transformations in substances and antioxidant activity induced by fermentation[23–25]. Fermentation concurrently facilitates the release and degradation of various substances, thus the effects often vary across different plant materials. For instance, the fermentation enhanced the release of phenolic compounds in pumpkin (Cucurbita maxima D.) silage, thereby mitigating the decline in antioxidant capacity resulting from a reduction in substances such as carotenoids[26]. On the contrary, after fermenting moringa, polyphenols were significantly reduced while accumulation of free amino acids and small peptides led to an increase in its antioxidant activity[27]. In this study, the additions of PP and LPl resulted in a significantly higher total flavonoids content compared to other groups, suggesting that PP and LPl demonstrated superior conversion/release efficiency for flavonoids during fermentation. The similar results were also found during the mulberry leaf fermentation[28]. Overall, the antioxidant activity increased after fermentation, in accordance with previous research on the fermentation of fruit and vegetable juices[29, 30]and tea[31, 32]. This improvement in antioxidant activity is believed to be attributed to the various enzymes produced by lactic acid bacteria during the fermentation process.
Furthermore, the antioxidant capacity of the LPl group was significantly higher than that of other groups in this study. The enhancement of antioxidant capacity varied among different strains of lactic acid bacteria, indicating that this process is highly strain-specific, likely associated with the inherent metabolic machinery of each strain[33]. Throughout the ensiling process, the intricate microbial community structure experiences intense competition, leading to diverse alterations in substances that collectively influence the antioxidant activity of alfalfa post-fermentation. Specific strains of lactic acid bacteria demonstrate heightened enzymatic production, releasing a greater quantity of flavonoids compounds and exhibiting a more efficient biotransformation capability.
Pearson correlation coefficient suggested that flavonoids content has close relationship with the antioxidant activity. Similar results were also observed in other studies, such as the yellowing process of rice[34] and the fermentation of seaweed by lactic acid bacteria[35]. For instance, the fermentation of Sargassum by lactic acid bacteria led to an increase in total flavonoids content and DPPH free radical scavenging activity[35]. To identify the specific compounds that play a key role in this process, further investigation using targeted metabolome analysis was performed to determine the type and content changes of flavonoids during fermentation.
Compared to natural fermentation, the addition of lactic acid bacteria resulted in distinctive biotransformation of flavonoids. The common substance in the lactic acid bacteria treatment group is 3,7,4 '- trihydroxyflavonoid, also known as resokaempferol, is a flavonoid compound with hydroxyl substitutions at positions 3, 7, and 4'. This compound has been demonstrated to possess the ability to scavenge DPPH free radicals and exhibit antibacterial activity[36, 37]. Additionally, it manifests excellent anti-inflammatory effects in vivo, contributing significantly to animal health[38]. Notably, the LPl group with Lactiplantibacillus plantarum exhibited distinct differences from control in terms of substance composition. This indicates that Lactiplantibacillus plantarum possessed a distinctive capability for flavonoids biotransformation during fermentation. For instance, adding LPl resulted in complete reduction of narcissin content. It is speculated that Lactiplantibacillus plantarum treatment induced high production of α-L-rhamnosidase, leading to glucoside bond cleavage within narcissin. Mueller et al. [39] assessed the hydrolytic capacity of 14 Lactobacillus strains on narcissin and other flavonoids and noted that rhamnosidase's capacity for flavonoids hydrolysis was highly specific to the strain. This specificity might explain why Lactiplantibacillus plantarum exhibited the most complete hydrolysis of narcissin. The characteristic flavonoid in the LPe group was identified as morusin, a kind of pyran-containing isopentenylated flavonoids. According to Pearson correlation coefficient, it was found that pedalitin, engeletin, 7-Methoxyisoflavone, 3,4'-Dihydroxyflavone, puerarin, 2'-Hydroxydaidzein were significantly negatively correlated with morusin (Fig. S2). Among them, pedalitin had the strongest negative correlation. Given that the pedalitin levels in the LPe group were markedly lower compared to other groups, its structure was subjected to analysis. It was speculated that under the influence of LPe, pedalitin undergoes a substitution reaction to yield morusin. In the study on the structure-activity relationship of flavonoids' antibacterial properties, Xie et al. [40] highlighted that isopentenylation enhanced the antibacterial efficacy of flavonoids. Hoi et al. [41] also confirmed the inhibitory effects of morusin extracted from Artocarpus nigrifolius on Bacillus subtilis and Staphylococcus aureus. The presence of this unique flavonoids may explain the near absence of Bacillus in the LPe group (Fig. S3). Acacetin, extracted from Glycyrrhiza glabra, has demonstrated potent DPPH radical scavenging activity [42]. In vivo experiments on mice [43], zebrafish [44] and other animals [45], acacetin has been confirmed to exert antioxidant capabilities. Various lactic acid bacteria strains demonstrated different abilities in biotransforming flavonoids [46].
Overall, high-molecular-weight flavonoids exhibited higher content in raw materials but lower content in each fermentation group. This suggests that the increase in the total content of alfalfa flavonoids after fermentation is not attributed to continued plant biosynthesis but is primarily a result of the release of bound flavonoids through microbial and enzymatic actions during fermentation. Simultaneously, high-molecular-weight flavonoids undergo degradation into smaller molecules and further engage in various biochemical reactions. For instance, a large amount of astragalin in the raw materials is deglucosided during fermentation and degraded into kaempferol. The degradation of luteolin to eriodictyol during microbial fermentation, Braune et al. [47] was also observed during the ensiling process in this study. Similarly, comparing samples before and after silage, a decrease in rutin levels and an increase in quercetin concentration were observed. This may be because certain microorganisms produce enzymes responsible for the degradation of rutin into quercetin under anaerobic conditions [48].