Chemical characteristics, antioxidant capacity, bacterial community, and metabolite composition of mulberry silage ensiling with lactic acid bacteria

Abstract

Mulberry has high crude protein and biologically active compounds but is difficult to be ensiled due to the lack of adequate epiphytic LAB. This study aimed to investigate the effects of inoculation with Lactiplantibacillus plantarum and Pediococcus pentosaceus isolated from mulberry with higher antioxidant capacity alone or in combination with Streptococcus bovis on chemical characteristics, antioxidant capacity, bacterial community, and metabolite composition of mulberry silage. The results showed that all inoculation groups had higher dry matter and lower pH than the control group, particularly in LP (dry matter, DM, 32.03% and pH = 4.44) and LP_PP_SB (DM, 31.68% and pH = 4.26) after 60 days of ensiling. Ammonia nitrogen (AN) content was the lowest in both LP_SB and LP_PP_SB groups, which were 1.86 g/kg FM and 1.05 g/kg FM, respectively, (P < 0.05). Only the LP_PP_SB group showed increased polyunsaturated fatty acids (PUFA, 1.2851 g/kg DM, P < 0.05) than the control group. Ferric-reducing antioxidant power (FRAP) values were increased in all inoculation-treated groups compared with the control group (P < 0.05). 2,2-Diphenyl-1-picrylhydrazyl (DDPH), 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and FRAP exhibited the highest levels in the LP_PP- and LP_PP_SB-treated groups. Enterobacter was dominant in both the control and SB-treated groups, and the relative abundance was 41.18% and 32.35%, respectively (P < 0.05). The relative abundance of Lactiplantibacillus was higher in the LP-, LP_PP-, and LP_SB-treated groups (81.84%−82.69%). Relative abundance of Pediococcus was higher in the PP-, PP_SB-, and LP_PP_SB-treated groups (74.27%−85.27%). Untargeted metabolomics analysis results showed that five flavonoids (apigenin, eriodictyol, quercetin-3-glucoside, rutin, and kaempferol-3-O-rutinoside)were upregulated in all inoculation groups (except for the SB-treated groups). Among them, eriodictyol was both positively correlated with ABTS and FRAP and also showed the highest relative abundance in the LP_PP- and LP_PP_SB-treated groups. To the best of our knowledge, this study was the first to investigate the relationship between inoculants of epiphytic lactic acid bacteria and antioxidant capacity by 16s rRNA Illumina sequencing technology and untargeted metabolomics analysis, respectively. Consequently, inoculated L. plantarum, P. pentosaceus alone, respectively, or in combination with S. bovis increased the relative abundance of Lactiplantibacillus and Pediococcus and decreased the relative abundance of Enterobacter, particularly in the LP_PP_SB-treated group. In addition, inoculants could increase the relative abundance of five flavonoids (apigenin, eriodictyol, quercetin-3-glucoside, rutin, and kaempferol-3-O-rutinoside), especially eriodictyol to improve the antioxidant capacity of mulberry silage.