Physiology of Industrially Relevant Bacteria: Freeze-drying tolerance of Lactobacillus reuteri and Pseudomonas chlororaphis and product formation of Lactococcus lactis metabolizing maltose

Sammanfattning: When the metabolic activity of bacteria is exploited by the food and biotechnology industries, freeze-drying is commonly used to produce concentrated bacterial cultures with enhanced storage stability. The freeze-drying tolerance of the probiotic strain Lactobacillus reuteri and the biocontrol agent Pseudomonas chlororaphis has been characterized in this work. Lb. reuteri was found to have higher inherent freeze-drying tolerance than P. chlororaphis. Glucose starvation and heat treatment conferred improved freeze-drying tolerance on both Lb. reuteri and P. chlororaphis, whereas chilling and NaCl stress did not. The highest freeze-drying survival rate of Lb. reuteri was observed when cells were harvested 2.5 h after the onset of glucose starvation in complex medium at pH 5. When Lb. reuteri was cultivated in a newly developed defined medium the freeze-drying survival rate was lower than after cultivation in complex medium, and addition of the compatible solutes glycine betaine and carnitine to the defined cultivation medium did not increase the freeze-drying tolerance. The freeze-drying medium was found to be important for the survival rate of both Lb. reuteri and P. chlororaphis, although the organisms required different compositions of the freeze-drying medium. In addition, the freeze-drying tolerance of P. chlororaphis was highly dependent on the cell concentration in the freeze-drying medium, with optimal survival rates in the interval 109–1010 cells per millilitre. The regulation of product formation was investigated for Lactococcus lactis metabolizing maltose. Fermentation end products, intracellular metabolites, inhibition of product-forming enzymes and the acidic proteome were analysed. More than 150 cytosolic protein spots were identified and quantified. L. lactis exhibits homolactic product formation during batch growth on glucose, while its growth on maltose results in mixed acid formation where formate, acetate and ethanol are formed in addition to lactate. The enzymes acetaldehyde/alcohol dehydrogenase (ADH) and phosphate acetyltransferase, involved in mixed acid product formation, were present at higher levels in maltose-grown cells than in glucose-grown cells. When L. lactis cells were resting, due to the absence of amino acids, the maltose consumption rate decreased three-fold compared with maltose-growing cells, and lactate was the only end product observed. The phenomenon of homolactic maltose fermentation of resting cells was observed in four L. lactis strains. The protein profiles of growing and resting L. lactis cells metabolizing maltose were to a high extent unaltered, even after several hours in the resting state. Glycolytic enzymes and enzymes involved in mixed acid product formation were present at similar levels in resting and growing cells metabolizing maltose, which indicates that regulation at enzyme level is predominant in resting cells. The intracellular concentrations of ADP, ATP and fructose 1,6-bisphosphate were elevated in resting cells compared with growing cells metabolizing maltose. The pool of ADP and ATP was sufficient to inhibit ADH, glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase, which might explain the changes in maltose consumption rate and product formation in resting cells. Maltose-metabolizing resting cells had lower levels of the 30S ribosomal proteins S1 and S2 than growing cells and the HPr protein was synthesized in a modified form, which is suggested to be due to misincorporation of alanine for valine.