Alfalfa as a vegetable source of β-carotene: The change mechanism of β-carotene during fermentation

The objectives of this study were to explore the β-carotene-producing bacteria and ascertain the main factors affecting β-carotene content via investigating the effects of various additives on β-carotene content, bacterial community succession, and quality of fermented alfalfa, using single-molecule real-time (SMRT) sequencing technology. Fresh alfalfa was fermented without (CON) or with squalene (SQ), the combination of Lactobacillus plantarum and cellulase (LPEN), and the combination of SQ and LPEN (SQLPEN) for 3, 45, and 90 d. The results showed that relative to the fresh alfalfa, extensive β-carotene loss in all groups occurred in the early fermentation phase (3 d) since epiphytic Pantoea agglomerans with the ability to produce β-carotene disappeared and β-carotene was oxidized by lipoxygenase and peroxidase. With the prolonged fermentation days, β-carotene content in all groups increased due to bacterial community succession in the middle and late phases of fermentation (45 and 90 d). The species L. parabuchneri, L. kunkeei, and L. kullabergensis (r = 0.591, 0.366, 0.341, orderly) had positive correlations with β-carotene content (P < 0.05). Bacterial functional potential prediction showed that species L. kunkeei, L. helsingborgensis, and L. kullabergensis had positive (r = 0.478, 0.765, 0.601) correlations with C10-C20 isoprenoid biosynthesis (P < 0.01), and L. helsingborgensis and L. kullabergensis had positive (r = 0.805, 0.522) correlations with β-carotene biosynthesis (P < 0.01). Additionally, the pH and propionic acid (r = -0.567, -0.504) had negative correlations with β-carotene content (P < 0.01). The CON group was preserved well after 90 d, LPEN and SQLPEN further improved fermentation quality. In conclusion, certain Lactobacillus had the potential for β-carotene biosynthesis, and high pH and propionic acid content were the unbenefited factors for β-carotene retention in fermented alfalfa.