Clarifying the relationships among bacteria, lipid-related enzymes, main polyunsaturated fatty acids and fat-soluble vitamins in alfalfa (Medicago sativa L.) silage using various sugar supplementations

The objective of this study was to clarify the relationships among bacteria, lipid-related enzymes, α-linolenic acid, α-tocopherol and β-carotene in alfalfa (Medicago sativa L.) silages, using without sugars as the control, and with 4 types of sugars (10 g kg⁻¹ fructose, 10 g kg⁻¹ glucose, 10 g kg⁻¹ sucrose, and 20 g kg⁻¹ molasses on fresh matter basis). After ensiling for 70 d, results showed that big losses of α-linolenic acid, α-tocopherol, and β-carotene were observed in the control silage. Supplementations of fructose, glucose, and sucrose increased the contents of α-linolenic acid and polyunsaturated fatty acids (P < 0.05) compared with the fresh alfalfa material. Similar to the control, there was a big loss of α-linolenic acid in the molasses-treated silage due to the high lipoxygenase activity and low activity ratio of lipase to lipoxygenase. Compared with the control, fructose supplementation reduced the losses of α-tocopherol and β-carotene (P < 0.05), and supplementations of glucose and molasses reduced the β-carotene loss after ensiling (P < 0.05). Next-generation sequencing results showed that Pediococcus (25.2 %), undesired Citrobacter (20.6 %), Lactococcus (17.7 %), and Weissella (17.2 %) rather than Lactobacillus (5.12 %) were predominant in the control silage. Molasses-treated silage had 48.1 % lower the abundance of Citrobacter (P < 0.05), and 255 % higher the abundance of Lactobacillus than the control silage (P < 0.05). Consequently, the highest lactic acid concentration and the lowest pH were observed in molasses-treated silage, indicating good fermentation quality. Person correlation analysis showed that Citrobacter (r = 0.84), Pediococcus (r = 0.73), and Lactococcus (r = 0.91) correlated with lipoxygenase positively, and correlated with α-linolenic acid and β-carotene negatively (r < -0.45); Lactobacillus (r = -0.56) and Leuconostoc (r = -0.48) correlated with α-tocopherol negatively. Redundancy analysis exhibited that Citrobacter, Pediococcus, and Lactococcus increased lipoxygenase to degrade α-linolenic acid and β-carotene and Lactobacillus involved in degrading α-tocopherol. In conclusion, sugar supplementations altered the bacterial community in alfalfa silage to reveal the relationships of Lactobacillus, Citrobacter, Pediococcus, and Lactococcus with lipoxygenase, α-linolenic acid, α-tocopherol, and β-carotene, which will provide deep thinking on the measures for reducing the losses of α-linolenic acid, α-tocopherol and β-carotene caused by above bacteria and lipid-related enzymes in alfalfa silage.