Properties of an acid-tolerant, persistent Cheddar cheese isolate, Lacticaseibacillus paracasei GCRL163

The distinctive flavours in hard cheeses are attributed largely to the activity of nonstarter lactic acid bacteria (NSLAB) which dominate the cheese matrix during maturation after lactose is consumed. Understanding how different strains of NSLAB survive, compete, and scavenge available nutrients is fundamental to selecting strains as potential adjunct starters which may influence product traits. Three Lacticaseibacillus paracasei isolates which dominated at different stages over 63-week maturation periods of Australian Cheddar cheeses had the same molecular biotype. They shared many phenotypic traits, including salt tolerance, optimum growth temperature, growth on N-acetylglucosamine and N-acetylgalactosamine plus delayed growth on D-ribose, carbon sources likely present in cheese due to bacterial autolysis. However, strains 124 and 163 (later named GCRL163) survived longer at low pH and grew on D-tagatose and D-mannitol, differentiating this phenotype from strain 122. When cultured on growth-limiting lactose (0.2%, wt/vol) in the presence of high concentrations of L-leucine and other amino acids, GCRL163 produced, and subsequently consumed lactate, forming acetic and formic acids, and demonstrated temporal accumulation of intermediates in pyruvate metabolism in long-term cultures. Strain GCRL163 grew in Tween 80-tryptone broths, a trait not shared by all L. casei-group dairy isolates screened in this study. Including citrate in this medium stimulated growth of GCRL163 above citrate alone, suggesting cometabolism of citrate and Tween 80. Proteomic analysis of cytosolic proteins indicated that growth in Tween 80 produced a higher stress state and increased relative abundance of three cell envelope proteinases (CEPs) (including PrtP and Dumpy), amongst over 230 differentially expressed proteins