A physiological comparative study of acid tolerance of Lactobacillus plantarum ZDY 2013 and L. plantarum ATCC 8014 at membrane and cytoplasm levels

Abstract

This study aimed to disclose the acid tolerance mechanism of Lactobacillus plantarum by comparing L. plantarum ZDY 2013 with the type strain L. plantarum ATCC 8014 in terms of cell membrane, energy metabolism, and amino acid metabolism. L. plantarum ZDY 2013 had a superior growth performance under acidic condition with 100-fold higher survival rate than that of L. plantarum ATCC 8014 at pH 2.5. To determine the acid tolerance physiological mechanism, cell integrity was investigated through scanning electron microscopy. The study revealed that L. plantarum ZDY 2013 maintained cell morphology and integrity, which is much better than L. plantarum ATCC 8014 under acid stress. Analysis of energy metabolism showed that, at pH 5.0, L. plantarum ZDY 2013 enhanced the activity of Na⁺/K⁺-ATPase and decreased the ratio of NAD⁺/NADH in comparison with L. plantarum ATCC 8014. Similarly, amino acid metabolism of intracellular arginine, glutamate, and alanine was improved in L. plantarum ZDY 2013. Correspondingly, the activity of arginine deiminase and glutamate decarboxylase of L. plantarum ZDY 2013 increased by 1.2-fold and 1.3-fold compared with L. plantarum ATCC 8014 in acid stress. In summary, it is demonstrated that the special physiological behaviors (integrity of cell membrane, enhanced energy metabolism, increased amino acid and enzyme level) of L. plantarum ZDY 2013 can protect the cells from acid stress.

Introduction

Lactic acid bacteria (LAB) have been used to produce fermented food over the past decades and have been developed as probiotics, which are generally recognized as safe (GRAS) (De Vries et al. 2006), for their health-promoting functions in the human gastrointestinal tract (Duary et al. 2010). Probiotics must maintain viability in the gut at a high concentration (at least 10⁶ cfu/mL) to be beneficial to the human host (Ferrando et al. 2015). Therefore, the stronger resistance of LAB strains against various extreme environments is a prerequisite for the development of probiotic supplements.

LAB are exposed to various stress conditions, such as acid, temperature, osmotic stress, or freeze drying in fermented food as well as in the gastrointestinal tract (Guchte et al. 2002). The tolerance of LAB to these stressors is critical for screening potential candidates for LAB application. As probiotic candidates, bacteria have to survive under extreme acidic conditions or digestion by various enzymes in the entire gastrointestinal tract (Wall et al. 2007). Given that acid stress is a pivotal issue for microbial survival, acid tolerance is generally considered as one of the criteria for selection of potential probiotics (De Angelis and Gobbetti 2004; Parvez et al. 2006).

Few publications have focused on the acid tolerance mechanism of LAB such as Lactococcus lactis (Rallu et al. 2000), Lactobacillus bulgaricus (Hernandez-Hernandez et al. 2012), Lactobacillus casei (Broadbent et al. 2010), Lactobacillus plantarum (Hamon et al. 2014), and Lactobacillus reuteri (Teixeira et al. 2014) or on the aspect of proteomic analysis level. More routinely studied are the acid tolerance, H⁺-ATPase proton pump, glutamate decarboxylase (GAD) system, and arginine deiminase (ADI) system of bacteria involved in pH homeostasis in Gram-positive bacteria (Cotter and Hill 2003). For example, the activity of H⁺-ATPase increases under acidic conditions to support the acid tolerance of Bacillus spp. (Shobharani and Halami 2014); the GAD system contributes to acid tolerance of Listeria monocytogenes in gastric fluid (Cotter et al. 2001); and ADI protects Bacillus cereus ATCC14579 (Senouci-Rezkallah et al. 2011), Escherichia coli (Lin et al. 1995), and L. monocytogenes (Lin et al. 1995; Ryan et al. 2009) against acid stress through the production of ammonia. On the other hand, alterations in cell membrane and metabolic pathways (e.g., energy metabolic pathway and amino acid metabolism) also control acid tolerance (Cotter and Hill 2003). For instance, an increased proportion of long-chained, monounsaturated fatty acids were produced in Streptococcus mutans UA159 in response to acid stress (Fozo and Quivey 2004); NADH oxidase, an enzyme responsible for oxidative stress, and which could possibly contribute to acid resistance, its activity of L. lactis (De Felipe et al. 1998), and Lactobacillus delbrueckii subsp. bulgaricus (Marty-Teysset et al. 2000) increased under acidic conditions; addition of glutamine increased the survival rate of L. reuteri 100–23 at pH 2.5 (Teixeira et al. 2014). To our best of our knowledge, few literature reports have disclosed information on the physiological changes of LAB due to acid stress, especially in L. plantarum.

Among a variety of LAB species, L. plantarum is one of the most widely used starters or probiotics in many products (Brinques and Ayub 2011). In our previous study, a strain of L. plantarum ZDY 2013 was proven to be an ideal probiotic candidate because of its tolerance to the extreme environment of the gastrointestinal tract. Moreover, L. plantarum ZDY 2013 underwent a global acid tolerance reaction in terms of mRNA levels (Huang et al. 2015), indicating that some unknown physiological changes takes place under extreme acidic conditions. To explore the acid stress mechanisms of L. plantarum ZDY 2013 through a physiological response, we used the type strain of L. plantarum ATCC 8014 as a standard. In the present study, we investigated the acid tolerance ability, cell integrity, and changes in energy and amino acid metabolism based on the different acid stress responses of L. plantarum ZDY 2013 and L. plantarum ATCC 8014.

Materials and methods

Bacteria strains, media, and growth conditions

L. plantarum ZDY 2013, isolated from a traditional fermented acid beans in our previous study (Huang et al. 2015), and a type strain, L. plantarum ATCC 8014, were used in this study. Both strains were incubated in MRS broth (Beijing Solarbio Science & Technology, Beijing, China) at 37 °C for 24 h under anaerobic condition. Growth conditions in MRS were applied in all subsequent experiments. For acid tolerance analyses, the pH of MRS broth was adusted to 6.2, 4.5 and 3.5.

Acid tolerance analyses and acid treatment

Growth of both L. plantarum strains was assessed under different acid conditions (pH 6.2, 4.5 and 3.5) in MRS broth, and the optical density (OD) at 600 nm was monitored at different time points using a microplate reader (VersaMaxTM Tunable microplate reader; Molecular Devices, Sunnyvale, CA). Additionally, the survival of L. plantarum ZDY 2013 was evaluated at pH 2.5 for 1.0 h in 0.01 M PBS with L plantarum ATCC 8014 for comparison.

Cells of L. plantarum ZDY 2013 and L. plantarum ATCC 8014 at mid-exponential growth phase were centrifuged at 10,000 g for 1 min, and incubated in PBS (pH 3.5, 4.5 and 5.0) at 37 °C for 1.0 h. Then, the acid-treated and untreated cells were used for further study.

Scanning electron microscopy

The morphological structure of acid treated and untreated cells were examined using scanning electron microscopy (SEM). The samples were prepared as described by Bron et al. (2004), with some modifications. Briefly, cells were harvested by centrifugation at 5000 g for 10 min and fixed in 2.5% (v/v) glutaraldehyde for 4 h at 4 °C. Subsequently, the cells were washed three times with deionized water, then dehydrated with ethanol by using 30%, 50%, 70%, 80% and 90% ethanol for one time, successively, and finally with absolute ethyl ethanol for three times, and then freeze-dried. The samples were fixed on an aluminum foil and sprayed with gold.

Measurement of membrane permeability

Membrane permeability was measured using the β-galactosidase substrate o-nitrophenyl-β-d-galactopyranoside (ONPG) as a probe, which can be degraded by β-galacto-sidase to produce o-nitrophenol (yellow color) (Zhu et al. 2014). Cells were rinsed once by centrifugation (5000 g, 5 min) and resuspended in 0.01 M PBS (pH 7.4) to an OD₆₀₀ of 1.0, then ONPG was added to a final concentration of 1.5 mM. After incubation at 37 °C for 30 min, the cell suspension was monitored at 420 nm using a spectrophotometer (Precision and Scientific, Shanghai, China).

Measurement of Na⁺/K⁺-ATPase activity

The Na⁺/K⁺-ATPase assay was carried out using the Na⁺/K⁺-ATPase assay kit (Suzhou Comin Biotechnology. Suzhou, China) following the manufacturer’s protocol. The activity of the Na⁺/K⁺-ATPase was expressed in Na⁺/K⁺-ATPase catalyzing ATP and the formation of 1 μmol inorganic phosphorus per hour per milligram of total protein. Protein concentration was determined by the method of Coomassie Brilliant Blue staining, using bovine serum albumin as a standard protein.

Determination of intracellular NAD⁺/NADH ratio

Cellular metabolism was stopped by putting cell suspension into liquid nitrogen for 3 min then the content of intracellular NAD⁺/NADH was determined using the NAD⁺/NADH assay kit (Suzhou Comin Biotechnology) following the manufacturer’s protocol.

Determination of intracellular amino acids

For the extraction of intracellular amino acids, 20 mL stationary phase cells was treated under different acid conditions (pH 6.2, 4.5 and 3.5) in 0.01 M PBS broth at 37 °C for 1.0 h, harvested by centrifugation at 12,000 g for 10 min, washed twice and resuspended in 1 mL of 0.2 M PBS, then boiled for 15 min. Cell debris was discarded after the centrifugation (12,000 g, 10 min, 4 °C). The supernatants were treated at room temperature after adding 1 mL 10% TCA for 10 min. Then the mixture was centrifuged at 12,000 g for 10 min at 4 °C, and the supernatants were analyzed with amino-acid analyzer (Sykam, Munich, Germany).

Measurement of ADI activity

ADI activity was measured as described by De Angelis et al. (2002) with some modifications. Cells resuspended in 0.05 M Tris-HCl buffer (pH 7.5) were sonicated for 10 min with an ultrasonic cell disruptor (Xinzhi, Ningbo, China) and centrifuged at 10,000 g for 10 min at 4 °C; the supernatant, containing cell wall and cytoplasm, was collected and considered as enzyme extract.

The reaction mixture (2.5 mL) consisted of enzyme extract, 0.05 M arginine and 0.05 M acetate buffer (pH 5.5). Controls without enzyme extract were included. After incubation at 37 °C for 1 h, the reaction was stopped by adding 500 μL 2 M HCl, and the precipitate was removed by centrifugation (10,000 g for 10 min at 4 °C). The citrulline content of the supernatant was determined as described by Archibald (1944) 1 mL supernatant was added to 1.5 mL acid mixture of H₃PO₄-H₂SO₄ (3:1, v/v) and 250 μL diacetyl monoxime (1.5%, w/v), mixed, and boiled in the dark for 30 min. After cooling for 10 min, the absorbance was measured at 460 nm. The standard curve for citrulline was determined by applying the same procedure to five standard solutions of citrulline (0, 10, 20, 30 and 40 μg/mL). One unit of activity was defined as the amount of enzyme catalyzing the formation of 1 μmol citrulline per hour per milligram of total protein. Protein concentration in the enzyme preparation was determined as described previously.

Measurement of GAD activity

GAD activity was measured as previously described for L. lactis (Johnson et al. 1997; Xu et al. 2003) with minor modifications. Cells were re-suspended in 2 mL cold McIlvaine buffer (pH 4.7) combined with 0.1 mM pyridoxal phosphate and 1 mM mercaptoethanol, sonicated for 10 min and centrifuged at 10,000 g for 10 min at 4 °C; the supernatant was taken as GAD extract.

The reaction mixture consisted of enzyme extract, 10 mM l-glutamate (pH 4.8) and 0.1 mM pyridoxal phosphate. Controls without enzyme extract were included. After incubation at 30 °C for 10 h, the reaction was stopped by addition of 200 μL 0.2 M sodium borate buffer, 1 mL 6% phenol solution and 400 μL sodium hypochloride coupled with ice water bath. Color development was carried out in boiling water for 10 min, then immediately put it in an ice water bath for 20 min. The optical density was read at 630 nm. The standard curve for γ-amino butyric acid (GABA) was determined by applying the same procedure to five standard solutions of GABA (0, 2, 4, 6 and 8 mM GABA). One unit of the activity was defined as the amount of enzyme that catalyzed the formation of 1 μmol GABA per hour per milligram of total protein. Protein concentration in the enzyme preparation was determined as described previously.

Statistical analysis

All results were expressed as mean ± standard deviation (SD). Statistical analysis was performed using two-way ANOVA procedure of SPSS 13.0 software (SPSS, Chicago, IL). Data was considered statistically significant when P < 0.05.

Results

Growth curve and acid tolerance ability of L. plantarum ZDY 2013 and ATCC 8014

To analyze the mechanism of acid tolerance of different L. plantarum, the growth curve of L. plantarum ZDY 2013 and ATCC 8014 in standard or acid conditions, and their cell survival in extreme acidic environment was investigated. At the initial pH of 6.2 (Fig. 1a), no significant difference between two strains was observed (P > 0.05). However, the biomass of L. plantarum ZDY 2013 was 32% higher than that of L. plantarum ATCC 8014 from 10 h when the initial pH was 4.5 (Fig. 1a). The growth of L. plantarum ATCC 8014 was severely inhibited at pH 3.5 (Fig. 1a), whereas L. plantarum ZDY 2013 grew well and exhibited a more than two-fold biomass increase. This finding clearly showed that L. plantarum ZDY 2013 had superior growth performance in the acidic condition. As shown in Fig. 1b, the survival rate of both cells decreased in PBS at pH 2.5. When exposed to acid stress for 1 h, the viable cells reached 1.9 × 10⁶ cfu/mL in L. plantarum ZDY 2013, which was more than 2 logs higher than that of L. plantarum ATCC 8014.

Fig. 1a,b

Tolerance analyses of Lactobacillus plantarum ZDY 2013 and L. plantarum ATCC 8014 cells under acid stress. a Growth of the two strains was calculated by measuring OD₆₀₀ in MRS at pH 6.2, 4.5, or 3.5. b The cell count reduction was calculated by log (N/N₀); N represented the survival counts, whereas N₀ represented the initial cell counts under acid stress of pH 2.5. All tests were performed in triplicate

Differentiation of cell integrity of L. plantarum strains under acid stress

Cell morphology of L. plantarum ZDY 2013 and ATCC 8014 was investigated by SEM (Fig. 2). Cells grown under standard conditions exhibited a rod-shaped, smooth-surface morphology in both L. plantarum ZDY 2013 and ATCC 8014 (Fig. 2a,c). When exposed to pH 3.5 for 1 h, the cells of L. plantarum ZDY 2013 showed a slight tendency to clump together, and their surfaces appeared to be less smooth, with some cavities (Fig. 2b). By contrast, the cells of L. plantarum ATCC 8014 showed significant changes under acidic environment; many cells appeared cracked (Fig. 2d), whereas L. plantarum ZDY 2013 retained the relative integrity of cell morphology at pH 3.5.

Fig. 2a–d

Morphological changes of Lactobacillus plantarum under acid stress as viewed by scanning electron microscopy (SEM). L. plantarum ZDY 2013 treated with MRS at a pH 6.2 or b pH 3.5 after 3 h; L. plantarum ATCC8014 treated with MRS at c pH 6.2 or d pH 3.5 after 3 h

To further compare the cell integrity of both strains, cell membrane permeability in series acid stress was evaluated by using ONPG as a substrate. As shown in Fig. 3, no difference in membrane permeability of either strain was found above pH 4.5 (P > 0.05). Nevertheless, L. plantarum ZDY 2013 presented significantly less membrane permeability at pH 3.5 when compared with L. plantarum ATCC 8014 (P < 0.001), again indicating that L. plantarum ZDY 2013 maintained cell morphology and integrity much better than L. plantarum ATCC 8014 under acid stress.

Fig. 3

Changes in membrane permeability of L. plantarum at different pH conditions. White bars L. plantarum ATCC 8014, gray bars L. plantarum ZDY 2013. All tests were performed in triplicate. Statistically significant difference was at P < 0.05

Na⁺/K⁺-ATPase

Na⁺/K⁺-ATPase is an integral membrane enzyme that plays a key role in maintaining electrochemical gradients. To investigate whether the Na⁺/K⁺-ATPase activity was related to the acid tolerance in L. plantarum, the activity of two strains was evaluated under different acid stress (Fig. 4). No obvious difference (P > 0.05) was found in the activity of Na⁺/K⁺-ATPase between L. plantarum ZDY 2013 and ATCC 8014 at either pH 6.2 or 4.5. However, the activity of L. plantarum ZDY 2013 and ATCC 8014 reached 8.2 ± 0.4 and 6.1 ± 0.4 U/mg, increasing by 1.88-fold and 1.62-fold at pH 5.0 when compared with pH 6.2. While exposed to pH 4.5, the activity was reduced to 6.4 ± 0.4 and 5.2 ± 0.4 U/mg, respectively.

Fig. 4

Effects of acid stress on the activity of Na⁺/K⁺-ATPase activity in L. plantarum. White bars L. plantarum ATCC 8014; gray bars L. plantarum ZDY 2013. All tests were performed in triplicate. Statistically significant difference was at P < 0.05

Intracellular NAD⁺/NADH ratio

Most energy in glucose metabolism is released by the respiratory chain, and the physiological change that occurs during respiration might be a consequence of the shift in the NAD⁺/NADH ratio (Lechardeur et al. 2011). In this study, the ratio of NAD⁺/NADH in L. plantarum ZDY 2013 and ATCC 8014 under acid stress was investigated (Fig. 5). At pH 6.2, pH 4.5 and pH 3.5, no significant difference was found in the ratio of NAD⁺/NADH of the two strains (P > 0.05). At pH 5.0, NAD⁺/NADH ratio in L. plantarum ZDY 2013 and L. plantarum ATCC 8014 increased to 6.8 ± 0.4 and 10.5 ± 1.2 (P < 0.01), respectively.

Fig. 5

Change in NAD⁺/NADH ratio in L. plantarum under acid stress. White bars L. plantarum ATCC 8014; gray bars L. plantarum ZDY 2013. All tests were performed in triplicate. Statistically significant difference was at P < 0.05

Amino acid changes

To investigate the effect of amino acid metabolism on acid tolerance mechanisms of L. plantarum, the chang in intracellular amino acid content of the two strains was monitored. Under standard conditions, no significant difference was found in arginine, glutamate, and alanine content (P > 0.05), whereas at pH 3.5, arginine content increased to 7.8 ± 0.8 and 3.9 ± 0.2 mg/mg (1.95-fold and 1.30-fold over pH 6.2) in L. plantarum ZDY 2013 and ATCC 8014, respectively (Fig. 6). L. plantarum ZDY 2013 maintained almost the same content of glutamate as in standard conditions at pH 4.5, whereas it decreased by 2.9-fold in L. plantarum ATCC 8014 (Fig. 6). A higher concentration of alanine accumulated in L. plantarum ZDY 2013 than in L. plantarum ATCC 8014 (Fig. 6). Before acid stress, the concentration of alanine was at 57.4 ± 6.7 and 42.1 ± 4.4 mg/mg in L. plantarum ZDY 2013 and ATCC 8014, respectively, and the level increased by 2.1-fold in the former but decreased by 0.7-fold in the latter. Proline and aspartate were detected in L. plantarum ZDY 2013 (2.6 ± 0.3 and 24.1 ± 2.8 mg/mg, respectively) but not in L. plantarum ATCC 8014 during acid stress. These results suggested that the accumulation of arginine, glutamate, and alanine might contribute to the acid tolerance response in L. plantarum ZDY 2013.

Fig. 6

Changes in intracellular arginine, glutamate, and alanine concentration L. plantarum ZDY 2013 and L. plantarum ATCC 8014. Concentration of intracellular arginine, glutamate, and alanine was determined. The unit of mg/mg indicates the amount of amino acids (mg) per milligram of protein. All tests were performed in triplicate. Statistically significant difference was at P < 0.05

ADI activity

The effect of pH on ADI activity was further determined in both strains. As shown in Fig. 7, at pH 5.0, ADI activity increased to 30.0 ± 1.8 and 25.09 ± 1.8 U/mg in L. plantarum ZDY 2013 and ATCC 8014, respectively. The activity decreased at pH below 5.0 in both strains, but no significant difference was found between them (P > 0.05) at pH 6.2. This result demonstrated that ADI was involved in the acid tolerance mechanism response in L. plantarum ZDY 2013.

Fig. 7

Arginine deiminase (ADI) activity of L. plantarum ZDY 2013 and L. plantarum ATCC 8014 under acid stress. All tests were performed in triplicate. Statistically significant difference was at P < 0.05

GAD activity

Acid stress might affect the GAD activity of cells of L. plantarum ZDY 2013 and ATCC 8014. As shown in Fig. 8, at pH 4.5, GAD activity of L. plantarum ZDY 2013 and ATCC 8014 increased by 3.3-fold and 2.7-fold, respectively, compared with that at pH 6.2. Although GAD activity increased in the two strains at pH 5.0, no significant difference was found between them (P > 0.05) at pH 6.2.

Fig. 8

Glutamate decarboxylase (GAD) activity of L. plantarum ZDY 2013 and L. plantarum ATCC 8014 under acid stress. White bars L. plantarum ATCC 8014, gray bars L. plantarum ZDY 2013. All tests were performed in triplicate. Statistically significant difference was at P < 0.05

Discussion

L. plantarum has a great potential to survive in acid stress environments either in vitro or in vivo; therefore, the acid tolerance properties of L. plantarum have attracted much attention because of its broad application in fermented food and probiotic supplements. In our previous study, L. plantarum ZDY 2013 was found to initiate a complex metabolic network involving cell membrane components, cellular metabolism, and energy production under acid stress, as reported at the molecular level by Huang et al. (2015). However, the related physiological mechanisms of the acid tolerance response are yet to be clarified. In this study, we performed a comparative and systematic study of two L. plantarum strains, namely, L. plantarum ZDY 2013 and L. plantarum ATCC 8014, and disclosed their acid tolerance response with regard to the cell membrane, energy metabolism, and amino acid metabolism.

Generally, the fluidity and integrity of the bacterial cytoplasmic membrane affects the viability of cells and their metabolic functions, particularly under stress conditions (Mykytczuk et al. 2007), since the cell envelope plays an important role in cellular growth, host defense, and in maintaining the stability of the intracellular environment (Wu et al. 2012). In our study, L. plantarum ZDY 2013 retained the relative integrity of cell morphology and the structure of intracellular membrane, which protects cells against damage in acid stress. On the other hand, a type strain, i.e., L. plantarum ATCC 8014, did not have the normal cell integrity of ZDY 2013 in acid stress; for instance, the survival rate of L. plantarum ZDY 2013 was approximately 100-fold higher than that of L. plantarum ATCC 8014 in pH 2.5 after 1 h.

Similar to many other microorganisms under acid stress, L. plantarum might start pH homeostasis activities including H⁺-ATPase proton pump, alterations in cell membrane, and metabolic pathways (Cotter and Hill 2003) to enhance its survival efficiency. Na⁺/K⁺-ATPase is a ubiquitous integral membrane enzyme (Towle 1984), which maintains the ion gradients of Na⁺ and K⁺ by consuming energy through ATP hydrolysis (Jutfelt 2006). In our study, we confirmed that L. plantarum ZDY 2013 strengthened proton cell pumping as supported by a 1.88-fold increase in Na⁺/K⁺-ATPase activities from an initial level that was significantly higher than that of L. plantarum ATCC 8014 in the same pH of 5.0 (P < 0.05). A similar result was reported also in L. reuteri (Lee et al. 2008). The activity of Na⁺/K⁺-ATPase decreased at pH 4.5. Wang et al. (2002) revealed that the active transport of Na⁺ and K⁺ was inhibited at pH 4.6–5.0 in their experiment. In our previous work, the gene transcription levels in ATP synthase (atpA and atpC) and glycerol-3-phosphate dehydrogenase (gspA) related to cellular energy metabolism were enhanced during acid stress (Huang et al. 2015).

Normally, NAD(H) plays an important role in cell death (Ying 2008), and can affect numerous enzymatic activities involved in the glycolysis pathway and tricarboxylic acid cycle (Fernie et al. 2004). At pH 5.0, the increased ratio of NAD⁺/NADH in L. plantarum ZDY 2013 and L. plantarum ATCC 8014 might relieve the inhibition of glyceraldehyde 3-phosphate dehydrogenase activity because a higher intracellular NAD⁺/NADH ratio could affect the enzymes using NAD or pyruvate as substrate (Lechardeur et al. 2011; Wendisch et al. 2006). An increased intracellular NAD⁺/NADH ratio and decreased levels of the content intracellular NAD⁺ and NADH could contribute to the increased of ATP/ADP ratio and enhance the energy metabolism (Lin et al. 2009).

Amino acid metabolism plays an important role in maintaining the homeostasis of intracellular pH, generating metabolic energy, and enhancing the cell resistance to environmental stress of LAB (Wu et al. 2013). In our work, we demonstrated that the arginine and alanine content of cells increased significantly for L. plantarum ZDY 2013 (P < 0.001), but was maintained or reduced for L. plantarum ATCC 8014 at pH 3.5. L. plantarum ZDY 2013 shifted the metabolic pathway by increasing the flux to arginine under acid conditions, and, based on arginine deiminase system, higher intracellular arginine concentration under acid conditions may be involved in acid tolerance in L. plantarum ZDY 2013. Glutamate content was maintained or decreased in both L. plantarum ZDY 2013 or L. plantarum ATCC 8014 at pH 4.5, respectively. Several previous research studies have demonstrated that many amino acids may protect cells against acid stress (Senouci-Rezkallah et al. 2011). Senouci-Rezkallah et al. (2011) investigated the effects of adding glutamate and arginine on acid tolerance of B. cereus ATCC 14579, and the results showed that cell survival at pH 4.0 increased by 1-log or 2-log populations, respectively.

Deiminase and decarboxylases were mainly involved in the acid stress response of LAB (Fernández and Zúñiga 2006). Our data revealed that, at pH 6.2 to 5.0, ADI activity increased and then decreased gradually until pH 3.5, but it was still relatively higher than the initial level. Arginine was converted to NH₃ in the ADI pathway, which contributed to the alkalization of cell cytoplasm, protecting it from acid stress damage (Lin et al. 1995; Ryan et al. 2009). Moreover, ADI was involved in the extrusion of cytoplasmic protons of B. cereus ATCC 14579 and regulation of intracellular pH of Lactobacillus fermentum IMDO 130101, helping cells to survive longer under acid stress (De Angelis et al. 2002; Senouci-Rezkallah et al. 2011). As for GAD, we proved that it was increased significantly (P < 0.05) until pH 4.5 and went down at the initial level. Fernández and Zúñiga (2006) reported that, at pH 4.7, optimal activity of GAD degraded Glu into GABA. Sanders et al. (1998) concluded that GAD activity of L. lactis was a particularly effective system for protecting cells from acid stress.

Conclusions

By comparing the morphology and survival ratio of L. plantarum ZDY 2013 and ATCC 8014 strains, the physiological behavior of their Na⁺/K⁺-ATPase and NAD⁺/NADH, amino acid content, and ADI and GAD activity, we might ascertain that the acid stress response of L. plantarum ZDY 2013 may be complicated. Together with our previous findings, further work should be done to determine the systematic regulative effect of amino acids on the acid tolerance of L. plantarum ZDY 2013.