Hydroxycinnamic Acids Used as External Acceptors of Electrons: an Energetic Advantage for Strictly Heterofermentative Lactic Acid Bacteria

Abstract

The metabolism of hydroxycinnamic acids by strictly heterofermentative lactic acid bacteria (19 strains) was investigated as a potential alternative energy route. Lactobacillus curvatus PE5 was the most tolerant to hydroxycinnamic acids, followed by strains of Weissella spp., Lactobacillus brevis, Lactobacillus fermentum, and Leuconostoc mesenteroides, for which the MIC values were the same. The highest sensitivity was found for Lactobacillus rossiae strains. During growth in MRS broth, lactic acid bacteria reduced caffeic, p-coumaric, and ferulic acids into dihydrocaffeic, phloretic, and dihydroferulic acids, respectively, or decarboxylated hydroxycinnamic acids into the corresponding vinyl derivatives and then reduced the latter compounds to ethyl compounds. Reductase activities mainly emerged, and the activities of selected strains were further investigated in chemically defined basal medium (CDM) under anaerobic conditions. The end products of carbon metabolism were quantified, as were the levels of intracellular ATP and the NAD ؉ /NADH ratio. Electron and carbon balances and theoretical ATP/glucose yields were also estimated. When CDM was supplemented with hydroxycinnamic acids, the synthesis of ethanol decreased and the concentration of acetic acid increased. The levels of these metabolites reflected on the alcohol dehydrogenase and acetate kinase activities. Overall, some biochemical traits distinguished the common metabolism of strictly heterofermentative strains: main reductase activity toward hydroxycinnamic acids, a shift from alcohol dehydrogenase to acetate kinase activities, an increase in the NAD ؉ /NADH ratio, and the accumulation of supplementary intracellular ATP. Taken together, the above-described metabolic responses suggest that strictly heterofermentative lactic acid bacteria mainly use hydroxycinnamic acids as external acceptors of electrons.

Metabolism of Fructophilic Lactic Acid Bacteria Isolated from the Apis mellifera L. Bee Gut: Phenolic Acids as External Electron Acceptors

“…Differences in phenotype during growth in GYP supplemented with p-coumaric acid at 2 mmol liter Ϫ1 were monitored using OmniLog PM technology (Biolog) as described above. The NAD ϩ /NADH ratio was determined as described by Filannino et al (39). Cells were recovered through centrifugation (10,000 ϫ g for 10 min at 4°C) and washed in cold phosphate-buffered saline (Sigma, St. Louis, MO).…”

“…Metabolism of phenolic acids during growth in FYP broth. FYP broth was supplemented with p-coumaric, caffeic, syringic, or gallic acid at a concentration of 1 mmol liter Ϫ1 (39,49,50). Cells from an overnight culture were inoculated (5% [vol/vol]) into supplemented FYP medium and incubated for 24 h at 30°C.…”

“…The MICs of phenolic acids (p-coumaric, caffeic, syringic, and gallic acids) during the growth of FLAB were determined by the critical dilution assay (39,49). Phenolic compounds were dissolved in methanol and diluted in FYP broth to a final concentration of 50 mmol liter Ϫ1 .…”

“…p-Coumaric acid was chosen as the substrate since it is the main phenolic acid present in pollens and induced the highest percentages of degradation by FLAB strains (33,35). The concentration of 2 mmol liter Ϫ1 matched conditions employed in previous investigations (39) and was well below the MIC of these phenolic acids against FLAB. Differences in phenotype during growth in GYP supplemented with p-coumaric acid 2 mmol liter Ϫ1 were monitored using the OmniLog phenotype microarray (PM) technology (Biolog) ( Fig.…”

“…Similarly to plant-associated bacteria (37), FLAB colonizing honeybees' GITs might have modulated specific molecular defense mechanisms to ensure the tolerance against the broad range of plant secondary metabolites ingested by bees as part of their diet. To the best of our knowledge, the adaptation to and metabolism of phenolic acids have been investigated exclusively for Lactobacillus plantarum and lactic acid bacteria used for wine making (e.g., Oenococcus oeni) and rarely for other Lactobacillus spp., Weissella spp., and Leuconostoc mesenteroides (38,39). To date, the metabolism of phenolic acids by FLAB has not yet been investigated, and more in general, FLAB remain an unexplored group.…”