A Systems-Wide Analysis of Proteolytic and Lipolytic Pathways Uncovers The Flavor-Forming Potential of The Gram-Positive Bacterium Macrococcus caseolyticus subsp. caseolyticus

Macrococcus caseolyticus subsp. caseolyticus is a Gram-positive, commensal organism documented to be present as a component of the secondary microflora in fermented foods such as Ragusano and Fontina cheeses and Cantonese sausage. In these products, the organism appears to play a role in ripening and the development of the final organoleptic qualities. However, the role of this organism in flavor generation is not well understood. Therefore, the objective of this study was to investigate the role of M. caseolyticus subsp. caseolyticus in flavor compound formation through an examination of enzymatic, metabolomic and genomic data. A bank of M. caseolyticus subsp. caseolyticus strains derived from a variety of niches were examined. Enzyme activities analyzed comprised those of the proteolytic and lipolytic cascades including cell-envelope proteinase (CEP), peptidases, esterases, lipases, aminotransferases and glutamate dehydrogenase (GDH). Strain to strain variation was observed, often associated with niche. All strains, except those isolated from non-dairy sources, demonstrated high CEP activity. Such high CEP activity associated with dairy strains implies the importance of this characteristic in the adaptation of these strains to a dairy-specific niche. However, limited downstream peptidolytic activity, in addition to a limited ability to generate free amino acids (FAA) was observed across all strains, indicating weak ability of this organism to generate amino-acid derived flavor compounds. Interestingly, the strains with high CEP activity also demonstrated high esterase activity and gas chromatography-mass spectrometry (GC-MS) analysis of the volatile compounds produced when these strains were grown in lactose-free milk demonstrated differences in the range and types of volatiles produced. In contrast to this metabolic versatility, comparative genome analysis revealed the distribution of components of the proteolytic and lipolytic system in these strains to be conserved. Overall, this study demonstrates the potential of M. caseolyticus subsp. caseolyticus to generate diverse volatile flavor compounds. Additionally, the identification of the highly active strain-specific cell wall bound caseolytic proteases deriving extensive casein hydrolysis, serves as a promising avenue which can be potentially harnessed in the future to produce greater and more diverse flavor compounds.