Effects of Lactic Acid Bacteria Fermentation on Calcium Release and Metabolites in Chicken Bone Paste

The effects of Lactobacillus acidophilus CICC6074 (LA), Limosilactobacillus reuteri WQ-Y1 (LR) and Lactiplantibacillus plantarum A3 (LP) on calcium release and metabolite composition during the fermentation of chicken bone paste were studied using chicken bone as raw materials. The growth characteristics of lactic acid bacteria (LAB) strains, pH changes, total acidity variations, calcium distribution and calcium-to-phosphorus ratio, composition and content of metabolites were investigated to reveal the effect of the growth of LAB on calcium release during the fermentation of chicken bone paste. Principal component analysis (PCA), correlation analysis, and KEGG metabolic pathway analysis were further employed to identify the key metabolic pathways during the fermentation of chicken bone paste. The results showed that, LA, LR and LP strains were able to effectively utilize chicken bone paste for growth and proliferation, and the maximum viable cell count was observed at 30 h during the fermentation processing. The total acidity contents of LA, LP and LR groups were 5.60, 3.76, and 3.75 g/L respectively, significantly higher total acidity content was shown in LA group compared to other treatment groups (P<0.05). Calcium release analysis revealed that the total calcium content increased from 181.33 mg/kg of control group to 1176.67 mg/kg of LR, 1310.00 mg/kg of LP, and 1916.67 mg/kg of LA, respectively. The free calcium content of LA, LP and LR groups was 40.60, 50.19, and 74.62 fold-time higher than that of control group, and the free calcium content of LA group was significantly higher compared to LP and LR groups (P<0.05). X-ray diffraction and infrared spectroscopy results indicated that hydroxyapatite of bone paste mainly existed in an amorphous form. The intensity of characteristic peaks of hydroxyapatite at 2926 and 1050 cm⁻¹ significantly decreased among LR, LP and LA groups compared to control group. LC-MS/MS analysis demonstrated that 37 metabolites were identified in the fermented bone paste. Principal Component Analysis (PCA) and correlation analysis revealed that lactic acid, pyruvate, sucrose, L-serine, and 5'-CMP were the key metabolites of fermented bone paste. KEGG metabolic pathway analysis indicated that pyrimidine metabolism, arginine and proline metabolism, glycolysis, pyruvate metabolism, taurine and hypotaurine metabolism, and tricarboxylic acid (TCA) cycle were the main metabolic pathways associated with lactic acid bacteria growth and calcium release during the fermentation of bone paste.