The metabolite set enrichment and metabolic pathway analyses were performed using MetaboAnalyst 4.0 to find out the most altered metabolic pathways following incubation in prolonged cold environment. The pathway impact value was calculated from topology pathway analysis, those with score higher than 0.10 was considered as a significant metabolic pathway. The analysis identified 10 matched pathways, including purine metabolism, pyrimidine metabolism, TCA cycle, butanoate metabolism, propionate metabolism, pyruvate metabolism, glycolysis and gluconeogenesis, fructose and mannose metabolism, pentose glucose interconversion, amino sugar and nucleotide sugar metabolism, sulfur metabolism, glyxolate decarboxylic metabolism, glycine serine and threonine metabolism and glycerolipid metabolism (Figure 4B). However, six metabolic pathways were significantly altered due to cold treatment, including TCA cycle, glycerolipid metabolism, pyrimidine metabolism, purine metabolism, peptidoglycan biosynthesis (Figure 4B). The quantitative enrichment analysis (MESA) was used to determine the major perturbed pathways affected in cold stressed samples (Figure 4A). Twenty biochemical pathways were identified by the analysis, seven of which were significantly affected by the cold treatment (p < 0.05) including citrate cycle, alanine-aspartate and glutamate metabolism, tyrosine metabolism, phenylalanine metabolism, propionate metabolism, butanoate metabolism, glyoxylate and decarboxylate metabolism (p < 0.05).