Integrated metabolomic analysis of Lactiplantibacillus plantarum NMGL2 reveals its survival and response to combinational cold and acidic conditions during storage of fermented milk

This study aimed to elucidate the molecular mechanism of Lactiplantibacillus plantarum NMGL2 (L. plantarum NMGL2) isolated from traditional Inner Mongolia cheese that how it survived under simultaneous cold and acid conditions, and developed potential protective strategies for bacterial survivability. L. plantarum NMGL2 was previously shown with numerous bioactivities such as antibiofilm, cholesterol-lowering effect, and bile acids binding capacity. In this study, non-directional UPLC-Q-TOF-MS/MS-based metabolomics was performed to investigate the metabolic variations in L. plantarum NMGL2 during refrigerated storage of the fermented milk to comprehend the underlying chemical process of bacterial resistance under the cold and acid conditions. Organic acids, fatty acids, polypeptides, amino acids, and carbohydrates were all among the 122 differently abundant metabolites discovered, which were mostly involved in organic acid, sugar, protein, amino acid, and purine metabolisms, This indicated that the bacteria counteracted the combinational coldness and acidity by increased biosynthesis of several important biomolecules such as glycolipids and glycoproteins for providing cell protection, glutamate for initiating cell deacidification process, and DNA for cell repairing, as well as cell membrane proteins and cell wall peptidoglycan. Western blot analysis confirmed up-regulation of PGRP protein, implicating that the cell wall peptidoglycan played a significant part in bacterial resistance under cold and acid conditions. Further SPME-GC/MS analysis demonstrated different volatile metabolites detected in the fermented milk during the storage, including 7 acids, 4 alcohols, 2 aldehydes, 4 ketones and 3 esters, which provided unique flavor characteristic of fermented milk for favorable consumer acceptance of the product. For the first time, our research elucidates the molecular mechanism by which L. plantarum survives simultaneous cold and acid conditions with development of potential protective strategies under such conditions.