Hexanoic acid addition helps to clarify the possible mechanisms of the increased β-carotene content during alfalfa fermentation

The objectives of this study were to evaluate the effect of hexanoic acid (HA) supplementations (0, as the control, CON; 0.05%, HA1; 0.1%, HA2; 0.2%, HA3) on β-carotene, and ascertain the way and key factors of HA influencing β-carotene content of alfalfa (Medicago sativa L.) after ensiled in an oxygen-free and dark conditions for 10, 40, and 80 d (from May to August, 2021). This was achieved by examining the dynamic change of β-carotene, activities of β-carotenerelated enzymes, and bacterial community succession of ensiled alfalfa, using operon crtNM identification, crtE gene quantitation, and single-molecule real-time sequencing technology. The results revealed that when compared with the fresh material, terminal alfalfa silage treated with different level of HA supplementations (0, 0.05, 0.1, 0.2%; fresh weight basis) increased β-carotene content up to 2.86, 85.8, 159, and 133%, accordingly. Meanwhile, alfalfa silage treated with higher levels of HA (0.1 and 0.2%) showed superior effects compared to those treated with lower levels of supplementation (0 and 0.05%). HA supplementation specifically facilitated the increase abundance of Lactobacillus kullabergensis and the emergence of L. senioris. Multiple linear regression models inferred that L. kullabergensis, L. apis, L. saniviri, L. senioris, peroxidase, phytoene desaturase, and lycopene β-cyclase positively regulated β-carotene. Conversely, Lactobacillus rennini and L. brevis adjusted β-carotene, negatively. Positive regulations of the above bacterial species and enzymes had a stronger role in increasing β-carotene than L. rennini and L. brevis. In conclusion, the β-carotene increase of ensiled alfalfa may be regulated by HA supplementation via multiple positive factors, including 4 special Lactobacillus species (L. kullabergensis, L. apis, L. saniviri, and L. senioris), and 3 vegetative β-carotene-related enzymes (peroxidase, phytoene desaturase, and lycopene β-cyclase).