Proteomes of subsp. LBB.B5 Incubated in Milk at Optimal and Low Temperatures.

We identified the proteins synthesized by subsp. strain LBB.B5 in laboratory culture medium (MRS) at 37°C and milk at 37 and 4°C. Cell-associated proteins were measured by gel-free, shotgun proteomics using high-performance liquid chromatography coupled with tandem mass spectrophotometry. A total of 635 proteins were recovered from all cultures, among which 72 proteins were milk associated (unique or significantly more abundant in milk). LBB.B5 responded to milk by increasing the production of proteins required for purine biosynthesis, carbohydrate metabolism (LacZ and ManM), energy metabolism (TpiA, PgK, Eno, SdhA, and GapN), amino acid synthesis (MetE, CysK, LBU0412, and AspC) and transport (GlnM and GlnP), and stress response (Trx, MsrA, MecA, and SmpB). The requirement for purines was confirmed by the significantly improved cell yields of subsp. when incubated in milk supplemented with adenine and guanine. The subsp. -expressed proteome in milk changed upon incubation at 4°C for 5 days and included increased levels of 17 proteins, several of which confer functions in stress tolerance (AddB, UvrC, RecA, and DnaJ). However, even with the activation of stress responses in either milk or MRS, subsp. did not survive passage through the murine digestive tract. These findings inform efforts to understand how subsp. is adapted to the dairy environment and its implications for its health-benefiting properties in the human digestive tract. subsp. has a long history of use in yogurt production. Although commonly cocultured with subsp. in milk, fundamental knowledge of the adaptive responses of subsp. to the dairy environment and the consequences of those responses on the use of subsp. as a probiotic remain to be elucidated. In this study, we identified proteins of subsp. LBB.B5 that are synthesized in higher quantities in milk at growth-conducive and non-growth-conductive (refrigeration) temperatures compared to laboratory culture medium and further examined whether those subsp. cultures were affected differently in their capacity to survive transit through the murine digestive tract. This work provides novel insight into how a major, food-adapted microbe responds to its primary habitat. Such knowledge can be applied to improve starter culture and yogurt production and to elucidate matrix effects on probiotic performance.