Introduction

The intracellular or cytoplasmic pH (pHin) is an essential factor for both metabolism and growth parameters due to its significant effect on different cellular processes during fermentation such as enzymatic functions, proton motive force, transport kinetics, and ATP generation (Hansen et al. 2016). In bacteria, the pHin is the relevant factor for most of the cell’s enzymatic machinery, and the pH gradient (ΔpH = pHex − pHin) across the cytoplasm membrane is the major component of the proton motive force in acidic medium (Kashket 1987; Hansen et al. 2016). The different methods used to determine the cytoplasmic pH of bacteria are generally divided into two classes: ion distribution methods such as flow dialysis and ion-selective electrodes and internal pH indicator methods such as 31P nuclear magnetic resonance spectroscopy (NMR) and fluorescent pH probes (Rottenberg 1979; Kashket 1985; Padan and Schuldiner 1986; Zhang et al. 2012; Wu et al. 2014; Bouix and Ghorbal 2015; Hansen et al. 2016; Kudo and Sasaki 2019; Xu et al. 2019). The fluorescent biosensors are generally divided into two groups: fluorescent dyes (probes) that possess several drawbacks including cytotoxic effects, limited cellular uptake, or leakage of the dye from cells and the genetically encoded fluorescent protein (FP)–based pH biosensors, which were developed to overcome some of the problems that are specifically associated with fluorescent dyes (Rupprecht et al. 2017). The radioactivity technique, e.g., accumulation of [14C] radioactive organic acid probe, was also reported for measuring intracellular pH in Lactococcus lactis subsp. cremoris (Mercade et al. 2000) and Leuconostoc oenos (Loubiere et al. 1992). Some of these methods are not available for every researcher because they depend on complex and expensive instruments such as NMR and radioactive and fluorometric methods.

Over the last two decades, the fluorescent probe method was proposed as a new and alternative method to measure the intracellular pH of lactic acid bacteria (Breeuwer et al. 1996; Glaasker et al. 1996; Marty-Teysset et al. 1996; Belguendouz et al. 1997; Cachon et al. 1998; Hache et al. 1999; Rault et al. 2009; Zhang et al. 2012; Wu et al. 2013, 2014; Bouix and Ghorbal 2015; Alwazeer et al. 2018; Kudo and Sasaki 2019). Given that different bacteria can exhibit a wide range of pHin values (typically from 5.6 to 9.0), it is impossible, in the fluorescent probe method, to employ a single pH probe which can cover a wide pH range because this method is limited to the range of pKa of probe ± 1 (Breeuwer et al. 1996; Glaasker et al. 1996; Bouix and Ghorbal 2015; Schäferling 2016), in addition to the dependence of the probe signal upon the esterase activity (Booth 1985; Kudo and Sasaki 2019).

The weak acid or base distribution methods are divided into separation methods by silicon oil centrifugation or filtration and non-separation methods such as flow dialysis and ion-selective electrodes (Rottenberg 1979). These methods are based on the assumption that the neutral lipophilic form of the probe (weak acid) is permeable across the cytoplasmic membrane and the ionized hydrophilic form is impermeable (Rottenberg 1979; Kashket 1985). The probe used must not be metabolized by the cells, neither bind to their components nor be actively transported, and it should be rapidly equilibrated (Rottenberg 1979). In these methods, the weak organic acids, especially the radiolabeled ones, are the most commonly used to measure the delta pH when the pHin is alkaline (Kashket 1985; McDonald et al. 1990). Non-separation of the cells from the external medium is generally recommended because this step might lead to leakage of the probe from the cells (Rottenberg 1979). Only the NMR, flow dialysis, and ion-selective electrode methods allow one to achieve this objective, but the first two methods require a relatively high cell concentration (c.a. 10 mg protein/mL) (Hellingwerf and van Hoorn 1985). Other advantages of the ion-selective electrode method are the simplicity of instruments and uses, instantaneous response time, and continuous monitoring (Rottenberg 1979; Hellingwerf and van Hoorn 1985).

In intracellular pH studies of lactic acid bacteria, the external acidic conditions are most commonly studied because these bacteria often grow in acidic food media. L. plantarum is a lactic acid bacterium that was widely used in the fermentation of vegetables, meat, and dairy products; and it was referenced as a major alteration factor in other food products such as citrus juices (Parish and Higgins 1988). In previous work, our team showed the advantages of ion-selective electrode method over the fluorescent one for the measurement of intracellular pH in Leuconostoc mesonteroides (Alwazeer et al. 2018). In this study, we describe the possibility of using the ion-selective electrode method as a novel application to study continuously the effects of environmental conditions, like medium pH (pHex), on the intracellular pH of living cells in Lactobacillus plantarum.

Materials and methods

Bacterial growth

The strain of L. plantarum used in this study was isolated from orange juice (Parish and Higgins 1988). It was cultivated in a modified MRS broth of pH 6.4 that did not contain tween 80 or acetate, and the cells were harvested in the exponential growth phase (determined spectrophotometrically by measuring the optical density of the growth medium) to avoid the stress conditions that may lead to the synthesis of the multidrug efflux proteins (Paulsen et al. 1996). The cells were washed twice in a 100 mmol/L potassium phosphate buffer (pH 7.0), energized by incubation with 0.5 mol/L glucose at 30 °C for 30 min. The cells were then harvested using a Beckman J2-HC centrifuge (Beckman Instruments, Inc., USA) with a JA-10 rotor for 5 min and 4420 g and concentrated at 18 mg protein/mL (Lowry et al. 1951) in a 100 mmol/L potassium phosphate buffer at appropriate pH. The replications were from independent growth cultures of different days.

Electrode system

A polyvinyl chloride (PVC) membrane sensitive to salicylate anions was developed in this study. This membrane was constructed as described previously (Setty et al. 1983) with some modifications introduced as follows: A solution (A) was prepared from 297 mg polyvinyl chloride (Sigma Chemical CO., St Louis, MO, USA) that dissolved slowly in a mixture of 0.7 mL of dioctylphtalate (Sigma Chemical CO., St Louis, MO, USA) and 6 mL of tetrahydrofuran (Sigma-Aldrich CO., Gillingham, England). A solution (B) was constituted from 100 mg tetraheptyl ammonium iodide (Merck CO., Schuchardt, Germany) dissolved in 2.4 mL tetrahydrophuran. The solution (B) was added to the solution (A) with vortexing for 20 min and then the mixture was poured into a glass Petri dish (9.5 cm diameter) that was previously washed with ethanol and tetrahydrophuran and dried. The closed Petri dish was placed on a horizontal surface at room temperature for 2 days (for slight evaporation). After the dehydration step, a round piece was punched out of the membrane film and placed on the electrode holder with an elastic ring. A filling solution of 2 mmol/L salicylate dissolved in a 100 mmol/L potassium phosphate buffer (pH 7.0) was added inside the electrode holder where there was a platinum electrode. The salicylate-selective electrode was equipped by a removable salicylate-selective membrane. This membrane was removed and changed before each new assay. The electrode readings were referenced to a standard calomel electrode (Radiometer Analytical S.A., Villeurbanne, France), and the voltage was passed through an electronic interface to the microcomputer (Fig. 1). The software used (Elit 8808, Ficher Bioblock, chemputrix, France) permitted a continuous measurement of salicylate concentration changes during the measuring tests. The medium pH was measured with a combined electrode (Inlab 427, Metler Toledo, S.A.R.L., Paris, France).

Fig. 1
figure 1

Schematic illustration for the measuring system of intracellular pH using the ion-selective electrode method. AH, a weak acid (salicylic acid)

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Measurement of intracellular pH

A small-scale bioreactor was used to determine the intracellular pH by using the ion-selective electrode method. The applied system contained a measuring vessel (Bioreactor) and an isolation system (to avoid electric interferences). The calibration curve was established to determine the initial concentration and the final concentration of salicylate anion, and the slope (S) was calculated. This procedure was repeated at the end of the experiment to check that the membrane was in working order during the experiment. The energized cells were added to the thermostated and stirred buffer at appropriate pH value, which contained 100 μmol/L of salicylate (the final concentration of cells was 1.8 mg protein/mL). The change in electrode response (ΔE) was recorded. A control assay was performed by adding 1 μmol/L of both ionophores (nigericin and valinomycin) to the concentrated and energized cells, and then the sample was incubated at 30 °C for 60 min before measurement. All the results in this work are the mean values for at least three measurements.

Calculations

The calculations were performed with the assumption that no probe binding occurred between the salicylate anions and the cellular proteins (i.e., binding constant Ki = 0) (Hellingwerf and van Hoorn 1985).

The dissociation constant (pKa) of salicylate equaled to 2.98; so, most of the probe was ionized at the studied pH values ranging from 4.0 to 7.0 and the concentration of the undissociated probe could be neglected (Hellingwerf and van Hoorn 1985). The intracellular pH calculations were performed according to equations reported in Alwazeer et al. (2018).

Results and discussion

Calibration curve

The probe used to measure the intracellular pH of microorganisms by the method of the weak acid–selective electrode must be rigorously chosen because it is known that the inhibitory action of weak acids increases with the decrease of both medium pH and Ka values. In the ion distribution method, benzoic acid (pKa = 4.20) was the probe most commonly used to measure the intracellular pH of acidophilic bacteria (Padan and Schuldiner 1986; Nannen and Hutkins 1991; Wouters et al. 1998). The use of this probe is limited at a value of pHex superior to 5.2 because it is recommended for the alkaline bacteria that the pKa of the probe should be inferior to the value of pHex − 1) (Rottenberg 1979). Booth (1985) reported that the acetic acid (pKa = 4.75), which could be used as a probe in the ion-selective electrode method for internal pH measurement, might be actively transported and might act as a carbon source in Escherichia coli, whereas benzoate acid showed interferences with metabolism. For all these reasons, salicylic acid that has a low pKa (2.98) was selected in the present study to achieve the pHin measurements. For salicylate concentrations ranging from 25 to 800 μmol/L, the electrode described in this work demonstrated a linear response range between 45 and 50 mV per decade change of concentration.

Effect of medium pH on intracellular pH

The effect of pH of the medium on the intracellular pH of L. plantarum was demonstrated in Fig. 2 and Table 1. When the medium pH decreases, the intracellular pH decreases, and the cells keep a cytoplasmic pH value higher than the external pH one. The ability of bacteria to maintain pH, i.e., homeostasis by membrane-bound primary proton pumps and solute-proton symport, would contribute to their growth and metabolic activities in acidic environments (Bouix and Ghorbal 2015). In this study, L. plantarum kept a neutral internal pH at acidic medium pH and the delta pH (ΔpH) ranged from 0.55 to 1.8 pH units at external pH ranged from 4.0 to 7.0. On the other hand, results showed that the delta pH was dissipated after adding the nigericin (Fig. 2B). The assumption that no binding occurs between the salicylate anions and the cellular proteins was proved by these observations because the internal salicylate anions left the cytoplasm to external medium after the addition of nigericin.

Fig. 2
figure 2

A Effect of pHex on the pHin in L. plantarum determined by the salicylate-selective electrode. The energized and concentrated cells were added to a 100 mmol/L potassium phosphate buffer at a cell concentration of 1.8 mg/mL protein. Symbols represent means ± standard deviations of three independent experiments. B The action of nigericin on the intracellular pH of L. plantarum suspended in a 100 mmol/L potassium phosphate buffer pH 5.0. The different treatments were effected as follows: a, cell addition at 1.8 mg/mL protein; b, nigericin addition at 1 μmol/L

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Table 1 Different methods used in the determination of pH intracellular in Lactobacillus plantarum
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It is important to indicate that the intracellular pH values of L. plantarum observed in this study were similar to those measured by other sophisticated methods such as radioactive probes and fluorescent probes (Table 1). Many researchers reported that the intracellular pH values measured by the weak acid distribution method were similar to those measured by the NMR method that was generally used to validate this method (weak acid–selective membrane) and also agreed with the results of the flow dialysis method (Hellingwerf and van Hoorn 1985; Kashket 1985). Furthermore, the difference in the bacterial strain can lead also to differences in the pH intercellular values for the same bacteria such the case of L. plantarum (Table 1) (Huang et al. 2016; Kudo and Sasaki 2019).

Conclusion

The important role of lactic acid bacteria in the food industry and food alteration has encouraged many researchers to study the energetic system of these bacteria whose intracellular pH is an important factor. Many types of research have been conducted to study the effect of environmental conditions on the intracellular pH of lactic acid bacteria such as medium pH, pressure, oxygen, and preservatives (Nannen and Hutkins 1991; Tseng et al. 1991; Cook and Russell 1994; Wouters et al. 1998). Many methods have been developed to meet this objective such as pH-sensitive dyes, 31P nuclear magnetic resonance, radioactively labeled weak acids, and fluorescent ratio imaging. The simplicity of the experimental protocol generally required to perform the measurements could make the ion-selective electrode method easily available for researchers compared with the complexity of instrumentation and the costly equipment requirement and time-consuming of the other methods. The application of radioactive salicylic acid as a probe in the determination of intracellular pH of L. plantarum showed the complexity of the procedure, the special precautions concerning the use of radioactive materials, and the high costs of instruments; these disadvantages could be avoided by the present ion-selective electrode method thanks to its simplicity, low costs, and reasonable safety conditions. Thus, this developed method could be of great interest to study the effects of acidic stress conditions on the intracellular pHin of bacteria at an external pH value as low as 4.0. In a previous study, we used the ion-selective electrode method to measure the delta psi (Δψ) of lactic acid bacteria (Hache et al. 1999), and in this study, we described the possibility of using the same system to measure the intracellular pH of one of those bacteria. Thus, now a single system can be used to measure, simultaneously, the effect of environmental conditions on both components of the proton motive force (Δψ) in bacteria.