Osmotic adjustments may exert benefits or drawbacks on Propionibacterium freudenreichii viability during freeze-drying.
Résumé
Propionibacterium freudenreichii is a beneficial bacterium consumed in cheeses and in probiotic food supplements. Indeed, selected strains of P. freudenreichii combine both technological and probiotic abilities. Selected strains were shown to adhere to intestinal cells and to modulate the mucosal immune response as well as the proliferation/apoptosis balance. P. freudenreichii are thus used as Swiss-type cheese starters and as probiotics in the industry. For these purposes, P. freudenreichii should be converted into a powdered form to be stabilized, without significant loss of viability. It suffers hyperosmotic constraints in different key steps of its use. This includes dry starter preparation, cheese making and transit through the human digestive tract. Osmotic adaptation, and the corresponding adjustments, are prerequisites to the use of P. freudenreichii. Moreover, during its large-scale industrial production, stabilisation of dried bacteria under a long-lasting live form is a critical point. This process being stressful, pre-treatments should aim at an enhanced tolerance. Culture media constitute a means to adapt bacteria in the aim to increase their survival during technological and digestive processes. In this study, we explored the impact of osmoadaptation in two P. freudenreichii strains, in different conditions, on survival.
Method
The maximal salt concentrations allowing growth, and inducing osmoadapatation, was selected, using a chemically defined medium. For all experiments, cells were collected at the beginning of the stationary phase. Accumulated osmoprotectants were identified and quantified by C and H NMR. Whole cell proteins were identified and quantified by LC-MS/MS. The different cultures were then submitted to oxidative, heat, acid, bile salts challenges and to freeze-drying.
Results
During growth, both P. freudenreichii strains accumulated trehalose, glutamate and glycine betaine. Osmoadaptation, strain-dependent, had different effects on multiple stress tolerance, depending on the presence of osmoprotectants. One of the two strains growth was restored by provided glycine betaine (GB). In this strain, salt stress preadaptation enhanced tolerance towards heat, oxidative, acid and freeze-drying challenges in the absence of GB. However, transport and accumulation of provided GB had deleterious effects on stress tolerance, while restoring optimal growth under hyperosmotic constraint. In the other strain, neither salt, nor GB, enhanced stress tolerance, which was constitutively low. Accordingly, whole cell proteomics revealed differences between strains, as well as different adaptive mechanisms triggered by salt in the presence and in the absence of GB.
Discussion
Osmotic adjustments may thus have positive or deleterious effects on industrial abilities of P. freudenreichii. Tuning of the culture medium composition allows driving bacterial adaptation and leads to better survival during freeze-drying and/or digestion. This, in turn, should lead to high yield and high-quality production of starters and probiotics.
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