Fermentation pH influences the physiological-state dynamics of Lactobacillus bulgaricus CFL1 during pH-controlled culture

Abstract

This study aims at better understanding the effects of fermentation pH and harvesting time on Lactobacillus bulgaricus CFL1 cellular state in order to improve knowledge of the dynamics of the physiological state and to better manage starter production. The Cinac system and multiparametric flow cytometry were used to characterize and compare the progress of the physiological events that occurred during pH 6 and pH 5 controlled cultures. Acidification activity, membrane damage, enzymatic activity, cellular depolarization, intracellular pH, and pH gradient were determined and compared during growing conditions. Strong differences in the time course of viability, membrane integrity, and acidification activity were displayed between pH 6 and pH 5 cultures. As a main result, the pH 5 control during fermentation allowed the cells to maintain a more robust physiological state, with high viability and stable acidification activity throughout growth, in opposition to a viability decrease and fluctuation of activity at pH 6. This result was mainly explained by differences in lactate concentration in the culture medium and in pH gradient value. The elevated content of the ionic lactate form at high pH values damaged membrane integrity that led to a viability decrease. In contrast, the high pH gradient observed throughout pH 5 cultures was associated with an increased energetic level that helped the cells maintain their physiological state. Such results may benefit industrial starter producers and fermented-product manufacturers by allowing them to better control the quality of their starters, before freezing or before using them for food fermentation.

FIG. 1.

Time course of L. bulgaricus CFL1 physiological characteristics throughout fermentations performed at pH 6. (a) NaOH consumption (—, g liter⁻¹) and rate of NaOH consumption (dNaOH/dt, - - -, g liter⁻¹ min⁻¹); (b) cultivability (▵, CFU ml⁻¹) and relative percentages of viable (•, %), dead (○, %), damaged (, %), and depolarized (+, %) cells; (c) specific acidification activity (tpH5.5, ▴, min); (d) intracellular pH (▪), pHext (□), and pH gradient (dpH = pHi - pHext) (░⃞). Results are the means of at least three independent measurements. They are expressed as a function of the time tm to reach the maximal rate of NaOH consumption as an adjusted time.

FIG. 2.

Time course of L. bulgaricus CFL1 physiological characteristics throughout fermentations performed at pH 5. (a) NaOH consumption (—, g liter⁻¹) and rate of NaOH consumption (dNaOH/dt, - - -, g liter⁻¹ min⁻¹); (b) cultivability (▵, CFU ml⁻¹) and relative percentages of viable (•, %), dead (○, %), damaged (, %); (c) specific acidification activity (tpH5.5, ▴, min); (d) intracellular pH (▪), pHext (□), and pH gradient (dpH = pHi - pHext) (░⃞). Results are the means of at least three independent measurements. They are expressed as a function of the time tm to reach the maximal rate of NaOH consumption as an adjusted time.

FIG. 3.

Relationship between the logarithm of dead cell counts measured by flow cytometry (D, cells ml⁻¹) and dissociated lactate concentration in the culture medium (Lac, g liter⁻¹), during fermentations performed at pH 6 (▪) and pH 5 (▵).

FIG. 4.

Schematic representation of physiological events during pH 6 or pH 5 L. bulgaricus CFL1 cultures. Bold lines correspond to main changes in the cellular state. Distinct cellular phases are numbered.