Metabolomic and microbial changes reveal the biotransformation of Calotropis gigantea into silage

Abstract Background: We recently reported that Calotropis gigantea could be used as a potential functional feed additive to specifically inhibit the detrimental rumen protozoa without impairing the fermentation traits. Meanwhile, to ensure the applicability at the farm level, bio-transforming Calotropis gigantea (giant milkweed, GM) into silage is of an utmost requisite which constitutes a long-term biological preservation. This study aimed at investigating the metabolite and microbiota profiles that can lead to the bio-transformation of Calotropis gigantea into silage, after supplementing fermentative bacteria and sucrose.Results: After ensiling, several metabolites like 3,4'-dihydroxybenzoic acid ethyl ester, 2-hydroxyethylphosphonic acid, 3,4'-dihydroxy-3',5'-dimethoxypropiophenone, vnilloylmalic acid, sedoheptulose, 2-hydroxy-3,5-dinitrobenzoic acid, L-arginine, putrescine, methyl linolenate and calactin were up-regulated while other like 2’-o-methyladenosine, xanthosine, 2-hydroxy-2-methyl propyl glucosinolate and isopentenyl adenine-7-N-glucoside were down-regulated making GM ensiling a biological process to manipulate the metabolite composition and structure for therapeutic needs. This was possible after the colonization by bacteria species like Bacteriodes salanitronis, B. plebeius, B. barnesiae, B. vulgatus, B. caecicola, Prevotella copri, Megamonas hypermegale, Olsenella sp. which increased in ensiling samples with Lactobacillus buchneri specifically found only in ensiled and inoculated samples. The "biosynthesis of secondary metabolites" was the KEGG pathway induced by the highest number of studied GM metabolites. PICRUSt2 identified the "brite hierarchies" as the more expressed microbial functional group and "human diseases and organismal systems" the least expressed one. Conclusion: These findings provide a fundamental description of the microbiota colonizing the plant GM for a successful ensiling process that induced a remarkable metabolomic changes. The cause and effect relationship predicted several metabolic pathways and the contribution of the microbiota profile to the biosynthesis of functional metabolites. Understanding the specific mechanisms modulated by the colonizing bacteria and fungi underpinning the bio-transformation into silage deserves further studies.