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

A physiological comparative study of acid tolerance of Lactobacillus plantarum ZDY 2013 and L.... Ann Microbiol (2017) 67:669–677 DOI 10.1007/s13213-017-1295-x ORIGINAL ARTICLE A physiological comparative study of acid tolerance of Lactobacillus plantarum ZDY 2013 and L. plantarum ATCC 8014 at membrane and cytoplasm levels 1 1 1 1 2 Yilin Guo & Ximei Tian & Renhui Huang & Xueying Tao & Nagendra P. Shah & 1,3 3 Hua Wei & Cuixiang Wan Received: 8 April 2017 /Accepted: 4 August 2017 /Published online: 23 August 2017 Springer-Verlag GmbH Germany and the University of Milan 2017 Abstract This study aimed to disclose the acid tolerance metabolism, increased amino acid and enzyme level) of mechanism of Lactobacillus plantarum by comparing L. plantarum ZDY 2013 can protect the cells from acid stress. L. plantarum ZDY 2013 with the type strain L. plantarum . . ATCC 8014 in terms of cell membrane, energy metabolism, Keywords Lactobacillus plantarum Acid tolerance Cell . . and amino acid metabolism. L. plantarum ZDY 2013 had a membrane Energy metabolism Amino acids 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 Introduction physiological mechanism, cell integrity was investigated through scanning electron microscopy. The study revealed Lactic acid bacteria (LAB) have been used to produce that L. plantarum ZDY 2013 maintained cell morphology fermented food over the past decades and have been de- and integrity, which is much better than L. plantarum veloped as probiotics, which are generally recognized as ATCC 8014 under acid stress. Analysis of energy metabo- safe (GRAS) (De Vries et al. 2006), for their health- lism showed that, at pH 5.0, L. plantarum ZDY 2013 en- promoting functions in the human gastrointestinal tract + + hanced the activity of Na /K -ATPase and decreased the (Duary et al. 2010). Probiotics must maintain viability in + 6 ratio of NAD /NADH in comparison with L. plantarum the gut at a high concentration (at least 10 cfu/mL) to be ATCC 8014. Similarly, amino acid metabolism of intracel- beneficial to the human host (Ferrando et al. 2015). lular arginine, glutamate, and alanine was improved in Therefore, the stronger resistance of LAB strains against L. plantarum ZDY 2013. Correspondingly, the activity of various extreme environments is a prerequisite for the de- arginine deiminase and glutamate decarboxylase of velopment of probiotic supplements. L. plantarum ZDY 2013 increased by 1.2-fold and 1.3-fold LAB are exposed to various stress conditions, such as acid, compared with L. plantarum ATCC 8014 in acid stress. In temperature, osmotic stress, or freeze drying in fermented food summary, it is demonstrated that the special physiological as well as in the gastrointestinal tract (Guchte et al. 2002). The behaviors (integrity of cell membrane, enhanced energy tolerance of LAB to these stressors is critical for screening potential candidates for LAB application. As probiotic candi- dates, bacteria have to survive under extreme acidic conditions or digestion by various enzymes in the entire gastrointestinal * Cuixiang Wan tract (Wall et al. 2007). Given that acid stress is a pivotal issue cuixiangwan@ncu.edu.cn for microbial survival, acid tolerance is generally considered as one of the criteria for selection of potential probiotics (De State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing Donglu, Nanchang, Jiangxi 330047, China Angelis and Gobbetti 2004;Parvez et al. 2006). Few publications have focused on the acid tolerance mech- Food and Nutritional Science, School of Biological Science, The University of Hong Kong, Pokfulam Road, Hong Kong, China anism of LAB such as Lactococcus lactis (Rallu et al. 2000), Lactobacillus bulgaricus (Hernandez-Hernandez et al. 2012), Jiangxi-OAI Joint Research Institute, Nanchang University, 235 Nanjing Donglu, Nanchang 330047, People’s Republic of China Lactobacillus casei (Broadbent et al. 2010), Lactobacillus 670 Ann Microbiol (2017) 67:669–677 plantarum (Hamon et al. 2014), and Lactobacillus reuteri type strain, L. plantarum ATCC 8014, were used in this study. (Teixeira et al. 2014) or on the aspect of proteomic analysis Both strains were incubated in MRS broth (Beijing Solarbio level. More routinely studied are the acid tolerance, H - Science & Technology, Beijing, China) at 37 °C for 24 h under ATPase proton pump, glutamate decarboxylase (GAD) system, anaerobic condition. Growth conditions in MRS were applied and arginine deiminase (ADI) system of bacteria involved in in all subsequent experiments. For acid tolerance analyses, the pH homeostasis in Gram-positive bacteria (Cotter and Hill pH of MRS broth was adusted to 6.2, 4.5 and 3.5. 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 analyses and acid treatment acid tolerance of Listeria monocytogenes in gastric fluid (Cotter et al. 2001); and ADI protects Bacillus cereus ATCC14579 Growth of both L. plantarum strains was assessed under dif- (Senouci-Rezkallah et al. 2011), Escherichia coli (Lin et al. ferent acid conditions (pH 6.2, 4.5 and 3.5) in MRS broth, and 1995), and L. monocytogenes (Lin et al. 1995; Ryan et al. the optical density (OD) at 600 nm was monitored at different 2009) against acid stress through the production of ammonia. time points using a microplate reader (VersaMaxTM Tunable On the other hand, alterations in cell membrane and metabolic microplate reader; Molecular Devices, Sunnyvale, CA). pathways (e.g., energy metabolic pathway and amino acid me- Additionally, the survival of L. plantarum ZDY 2013 was tabolism) also control acid tolerance (Cotter and Hill 2003). For evaluated at pH 2.5 for 1.0 h in 0.01 M PBS with L plantarum instance, an increased proportion of long-chained, monounsat- ATCC 8014 for comparison. urated fatty acids were produced in Streptococcus mutans Cells of L. plantarum ZDY 2013 and L. plantarum ATCC UA159 in response to acid stress (Fozo and Quivey 2004); 8014 at mid-exponential growth phase were centrifuged at NADH oxidase, an enzyme responsible for oxidative stress, 10,000 g for 1 min, and incubated in PBS (pH 3.5, 4.5 and and which could possibly contribute to acid resistance, its ac- 5.0) at 37 °C for 1.0 h. Then, the acid-treated and untreated tivity of L. lactis (DeFelipeet al. 1998), and Lactobacillus cells were used for further study. delbrueckii subsp. bulgaricus (Marty-Teysset et al. 2000)in- creased under acidic conditions; addition of glutamine in- creased the survival rate of L. reuteri 100–23 at pH 2.5 Scanning electron microscopy (Teixeira et al. 2014). To our best of our knowledge, few liter- ature reports have disclosed information on the physiological The morphological structure of acid treated and untreated changes of LAB due to acid stress, especially in L. plantarum. cells were examined using scanning electron microscopy Among a variety of LAB species, L. plantarum is one of (SEM). The samples were prepared as described by Bron et al. (2004), with some modifications. Briefly, cells were the mostwidelyusedstartersorprobioticsinmanyproducts (Brinques and Ayub 2011). In our previous study, a strain of harvested by centrifugation at 5000 g for10min andfixed L. plantarum ZDY 2013 was proven to be an ideal probiotic in 2.5% (v/v) glutaraldehyde for 4 h at 4 °C. Subsequently, candidate because of its tolerance to the extreme environment the cells were washed three times with deionized water, of the gastrointestinal tract. Moreover, L. plantarum ZDY then dehydrated with ethanol by using 30%, 50%, 70%, 2013 underwent a global acid tolerance reaction in terms of 80% and 90% ethanol for one time, successively, and fi- mRNA levels (Huang et al. 2015), indicating that some un- nally with absolute ethyl ethanol for three times, and then known physiological changes takes place under extreme acid- freeze-dried. The samples were fixed on an aluminum foil ic conditions. To explore the acid stress mechanisms of and sprayed with gold. 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 toler- Measurement of membrane permeability ance ability, cell integrity, and changes in energy and amino acid metabolism based on the different acid stress responses of Membrane permeability was measured using the β- L. plantarum ZDY 2013 and L. plantarum ATCC 8014. 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. Materials and methods 2014). Cells were rinsed once by centrifugation (5000 g, 5min)and resuspended in0.01MPBS(pH 7.4) toanOD Bacteria strains, media, and growth conditions 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 suspen- L. plantarum ZDY 2013, isolated from a traditional fermented sion was monitored at 420 nm using a spectrophotometer acid beans in our previous study (Huang et al. 2015), and a (Precision and Scientific, Shanghai, China). Ann Microbiol (2017) 67:669–677 671 + + Measurement of Na /K -ATPase activity cooling for 10 min, the absorbance was measured at 460 nm. The standard curve for citrulline was determined by applying + + + + The Na /K -ATPase assay was carried out using the Na /K - the same procedure to five standard solutions of citrulline (0, ATPase assay kit (Suzhou Comin Biotechnology. Suzhou, 10, 20, 30 and 40 μg/mL). One unit of activity was defined as China) following the manufacturer’s protocol. The activity the amount of enzyme catalyzing the formation of 1 μmol + + + + of the Na /K -ATPase was expressed in Na /K -ATPase cat- citrulline per hour per milligram of total protein. Protein con- alyzing ATP and the formation of 1 μmol inorganic phospho- centration in the enzyme preparation was determined as de- rus per hour per milligram of total protein. Protein concentra- scribed previously. tion was determined by the method of Coomassie Brilliant Blue staining, using bovine serum albumin as a standard Measurement of GAD activity protein. GAD activity was measured as previously described for Determination of intracellular NAD /NADH ratio L. lactis (Johnson et al. 1997; Xu et al. 2003) with minor modifications. Cells were re-suspended in 2 mL cold Cellular metabolism was stopped by putting cell suspension McIlvaine buffer (pH 4.7) combined with 0.1 mM pyridoxal into liquid nitrogen for 3 min then the content of intracellular phosphate and 1 mM mercaptoethanol, sonicated for 10 min + + NAD /NADH was determined using the NAD /NADH assay and centrifuged at 10,000 g for 10 min at 4 °C; the supernatant kit (Suzhou Comin Biotechnology) following the manufac- was taken as GAD extract. turer’s protocol. The reaction mixture consisted of enzyme extract, 10 mM L-glutamate (pH 4.8) and 0.1 mM pyridoxal phosphate. Determination of intracellular amino acids Controls without enzyme extract were included. After incuba- tion at 30 °C for 10 h, the reaction was stopped by addition of For the extraction of intracellular amino acids, 20 mL station- 200 μL 0.2 M sodium borate buffer, 1 mL 6% phenol solution ary phase cells was treated under different acid conditions and 400 μL sodium hypochloride coupled with ice water bath. (pH 6.2, 4.5 and 3.5) in 0.01 M PBS broth at 37 °C for Color development was carried out in boiling water for 1.0 h, harvested by centrifugation at 12,000 g for 10 min, 10 min, then immediately put it in an ice water bath for washed twice and resuspended in 1 mL of 0.2 M PBS, then 20 min. The optical density was read at 630 nm. The standard boiled for 15 min. Cell debris was discarded after the centri- curve for γ-amino butyric acid (GABA) was determined by fugation (12,000 g, 10 min, 4 °C). The supernatants were applying the same procedure to five standard solutions of treated at room temperature after adding 1 mL 10% TCA for GABA (0, 2, 4, 6 and 8 mM GABA). One unit of the activity 10 min. Then the mixture was centrifuged at 12,000 g for was defined as the amount of enzyme that catalyzed the for- 10 min at 4 °C, and the supernatants were analyzed with mation of 1 μmol GABA per hour per milligram of total amino-acid analyzer (Sykam, Munich, Germany). protein. Protein concentration in the enzyme preparation was determined as described previously. Measurement of ADI activity Statistical analysis ADI activity was measured as described by De Angelis et al. (2002) with some modifications. Cells resuspended in 0.05 M All results were expressed as mean ± standard deviation (SD). Tris-HCl buffer (pH 7.5) were sonicated for 10 min with an Statistical analysis was performed using two-way ANOVA ultrasonic cell disruptor (Xinzhi, Ningbo, China) and centri- procedure of SPSS 13.0 software (SPSS, Chicago, IL). Data fuged at 10,000 g for 10 min at 4 °C; the supernatant, contain- was considered statistically significant when P <0.05. ing cell wall and cytoplasm, was collected and considered as enzyme extract. The reaction mixture (2.5 mL) consisted of enzyme extract, Results 0.05 M arginine and 0.05 M acetate buffer (pH 5.5). Controls without enzyme extract were included. After incubation at Growth curve and acid tolerance ability of L. plantarum 37 °C for 1 h, the reaction was stopped by adding 500 μL ZDY 2013 and ATCC 8014 2 M HCl, and the precipitate was removed by centrifugation (10,000 g for 10 min at 4 °C). The citrulline content of the To analyze the mechanism of acid tolerance of different supernatant was determined as described by Archibald (1944) L. plantarum, the growth curve of L. plantarum ZDY 2013 1 mL supernatant was added to 1.5 mL acid mixture of and ATCC 8014 in standard or acid conditions, and their cell H PO -H SO (3:1, v/v) and 250 μL diacetyl monoxime survival in extreme acidic environment was investigated. At 3 4 2 4 (1.5%, w/v), mixed, and boiled in the dark for 30 min. After the initial pH of 6.2 (Fig. 1a), no significant difference 672 Ann Microbiol (2017) 67:669–677 between two strains was observed (P >0.05). However,the To further compare the cell integrity of both strains, cell biomass of L. plantarum ZDY 2013 was 32% higher than that membrane permeability in series acid stress was evaluated by of L. plantarum ATCC 8014 from 10 h when the initial pH using ONPG as a substrate. As shown in Fig. 3, no difference was 4.5 (Fig. 1a). The growth of L. plantarum ATCC 8014 in membrane permeability of either strain was found above was severely inhibited at pH 3.5 (Fig. 1a), whereas pH 4.5 (P > 0.05). Nevertheless, L. plantarum ZDY 2013 L. plantarum ZDY 2013 grew well and exhibited a more than presented significantly less membrane permeability at two-fold biomass increase. This finding clearly showed that pH 3.5 when compared with L. plantarum ATCC 8014 L. plantarum ZDY 2013 had superior growth performance in (P < 0.001), again indicating that L. plantarum ZDY 2013 the acidic condition. As shown in Fig. 1b, the survival rate of maintained cell morphology and integrity much better than both cells decreased in PBS at pH 2.5. When exposed to acid L. plantarum ATCC 8014 under acid stress. stress for 1 h, the viable cells reached 1.9 × 10 cfu/mL in + + L. plantarum ZDY 2013, which was more than 2 logs higher Na /K -ATPase than that of L. plantarum ATCC 8014. + + Na /K -ATPase is an integral membrane enzyme that plays a key role in maintaining electrochemical gradients. To investi- + + Differentiation of cell integrity of L. plantarum strains gate whether the Na /K -ATPase activity was related to the under acid stress acid tolerance in L. plantarum, the activity of two strains was evaluated under different acid stress (Fig. 4). No obvious dif- + + Cell morphology of L. plantarum ZDY 2013 and ATCC 8014 ference (P > 0.05) was found in the activity of Na /K -ATPase was investigated by SEM (Fig. 2). Cells grown under stan- between L. plantarum ZDY 2013 and ATCC 8014 at either dard conditions exhibited a rod-shaped, smooth-surface pH 6.2 or 4.5. However, the activity of L. plantarum ZDY morphology in both L. plantarum ZDY 2013 and ATCC 2013 and ATCC 8014 reached 8.2 ± 0.4 and 6.1 ± 0.4 U/mg, 8014 (Fig. 2a,c). When exposed to pH 3.5 for 1 h, the cells increasing by 1.88-fold and 1.62-fold at pH 5.0 when com- of L. plantarum ZDY 2013 showed a slight tendency to pared with pH 6.2. While exposed to pH 4.5, the activity was clump together, and their surfaces appeared to be less reduced to 6.4 ± 0.4 and 5.2 ± 0.4 U/mg, respectively. smooth, with some cavities (Fig. 2b). By contrast, the cells of L. plantarum ATCC 8014 showed significant changes Intracellular NAD /NADH ratio under acidic environment; many cells appeared cracked (Fig. 2d), whereas L. plantarum ZDY 2013 retained the Most energy in glucose metabolism is released by the respi- relative integrity of cell morphology at pH 3.5. ratory chain, and the physiological change that occurs during 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 OD in MRS at pH 6.2, 4.5, or 3.5. b The cell count reduction was calculated by log (N/N ); N represented the survival counts, whereas N represented the initial cell counts under acid stress of pH 2.5. All tests were performed in triplicate Ann Microbiol (2017) 67:669–677 673 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 respiration might be a consequence of the shift in the NAD / (P > 0.05), whereas at pH 3.5, arginine content increased NADH ratio (Lechardeur et al. 2011). In this study, the ratio of to 7.8 ± 0.8 and 3.9 ± 0.2 mg/mg (1.95-fold and 1.30-fold NAD /NADH in L. plantarum ZDY 2013 and ATCC 8014 over pH 6.2) in L. plantarum ZDY 2013 and ATCC 8014, under acid stress was investigated (Fig. 5). At pH 6.2, pH 4.5 respectively (Fig. 6). L. plantarum ZDY 2013 maintained and pH 3.5, no significant difference was found in the ratio of almost the same content of glutamate as in standard condi- + + NAD /NADH of the two strains (P > 0.05). At pH 5.0, NAD / tions at pH 4.5, whereas it decreased by 2.9-fold in NADH ratio in L. plantarum ZDY 2013 and L. plantarum L. plantarum ATCC 8014 (Fig. 6). A higher concentration ATCC 8014 increased to6.8 ±0.4 and10.5±1.2(P <0.01), of alanine accumulated in L. plantarum ZDY 2013 than in respectively. 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, Amino acid changes 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 To investigate the effect of amino acid metabolism on acid L. plantarum ZDY 2013 (2.6 ± 0.3 and 24.1 ± 2.8 mg/mg, tolerance mechanisms of L. plantarum, the chang in intra- respectively) but not in L. plantarum ATCC 8014 during acid cellular amino acid content of the two strains was moni- stress. These results suggested that the accumulation of argi- tored. Under standard conditions, no significant difference nine, glutamate, and alanine might contribute to the acid toler- was found in arginine, glutamate, and alanine content ance response in L. plantarum ZDY 2013. 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 <0.05 674 Ann Microbiol (2017) 67:669–677 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 ADI activity probiotic supplements. In our previous study, L. plantarum ZDY 2013 was found to initiate a complex metabolic network The effect of pH on ADI activity was further determined in involving cell membrane components, cellular metabolism, both strains. As shown in Fig. 7, at pH 5.0, ADI activity in- and energy production under acid stress, as reported at the creasedto30.0 ± 1.8and 25.09 ± 1.8U/mgin L. plantarum molecular level by Huang et al. (2015). However, the related ZDY 2013 and ATCC 8014, respectively. The activity de- physiological mechanisms of the acid tolerance response are creased at pH below 5.0 in both strains, but no significant yet to be clarified. In this study, we performed a comparative difference was found between them (P > 0.05) at pH 6.2. and systematic study of two L. plantarum strains, namely, This result demonstrated that ADI was involved in the acid L. plantarum ZDY 2013 and L. plantarum ATCC 8014, and tolerance mechanism response in L. plantarum ZDY 2013. disclosed their acid tolerance response with regard to the cell membrane, energy metabolism, and amino acid metabolism. GAD activity Generally, the fluidity and integrity of the bacterial cyto- plasmic membrane affects the viability of cells and their met- Acid stress might affect the GAD activity of cells of abolic functions, particularly under stress conditions L. plantarum ZDY 2013 and ATCC 8014. As shown in (Mykytczuk et al. 2007), since the cell envelope plays an Fig. 8, at pH 4.5, GAD activity of L. plantarum ZDY 2013 important role in cellular growth, host defense, and in main- and ATCC 8014 increased by 3.3-fold and 2.7-fold, respec- taining the stability of the intracellular environment (Wu et al. tively, compared with that at pH 6.2. Although GAD activity 2012). In our study, L. plantarum ZDY 2013 retained the increased in the two strains at pH 5.0, no significant differ- relative integrity of cell morphology and the structure of in- ence was found between them (P > 0.05) at pH 6.2. tracellular 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 Discussion 2013 in acid stress; for instance, the survival rate of L. plantarum ZDY 2013 was approximately 100-fold higher L. plantarum has a great potential to survive in acid stress than that of L. plantarum ATCC 8014 in pH 2.5 after 1 h. environments either in vitro or in vivo; therefore, the acid Similar to many other microorganisms under acid stress, tolerance properties of L. plantarum have attracted much at- L. plantarum might start pH homeostasis activities including tention because of its broad application in fermented food and H -ATPase proton pump, alterations in cell membrane, and 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 Ann Microbiol (2017) 67:669–677 675 Fig. 6 Changes in intracellular arginine, glutamate, and alanine determined. The unit of mg/mg indicates the amount of amino acids concentration L. plantarum ZDY 2013 and L. plantarum ATCC 8014. (mg) per milligram of protein. All tests were performed in triplicate. Concentration of intracellular arginine, glutamate, and alanine was Statistically significant difference was at P <0.05 metabolic pathways (Cotter and Hill 2003) to enhance its sur- phosphate dehydrogenase activity because a higher intracellu- + + + vival efficiency. Na /K -ATPase is a ubiquitous integral mem- lar NAD /NADH ratio could affect the enzymes using NAD brane enzyme (Towle 1984), which maintains the ion gradi- or pyruvate as substrate (Lechardeur et al. 2011; Wendisch + + + ents of Na and K by consuming energy through ATP hydro- et al. 2006). An increased intracellular NAD /NADH ratio lysis (Jutfelt 2006). In our study, we confirmed that and decreased levels of the content intracellular NAD and L. plantarum ZDY 2013 strengthened proton cell pumping NADH could contribute to the increased of ATP/ADP ratio + + as supported by a 1.88-fold increase in Na /K -ATPase activ- and enhance the energy metabolism (Lin et al. 2009). ities from an initial level that was significantly higher than that Amino acid metabolism plays an important role in of L. plantarum ATCC 8014 in the same pH of 5.0 (P <0.05). maintaining the homeostasis of intracellular pH, generat- A similar result was reported also in L. reuteri (Lee et al. ing metabolic energy, and enhancing the cell resistance to + + 2008). The activity of Na /K -ATPase decreased at pH 4.5. environmental stress of LAB (Wu et al. 2013). In our Wang et al. (2002) revealed that the active transport of Na work, we demonstrated that the arginine and alanine con- and K was inhibited at pH 4.6–5.0 in their experiment. In our tent of cells increased significantly for L. plantarum ZDY previous work, the gene transcription levels in ATP synthase 2013 (P < 0.001), but was maintained or reduced for (atpA and atpC) and glycerol-3-phosphate dehydrogenase L. plantarum ATCC 8014 at pH 3.5. L. plantarum ZDY (gspA) related to cellular energy metabolism were enhanced 2013 shifted the metabolic pathway by increasing the flux during acid stress (Huang et al. 2015). to arginine under acid conditions, and, based on arginine Normally, NAD(H) plays an important role in cell death deiminase system, higher intracellular arginine concentra- (Ying 2008), and can affect numerous enzymatic activities tion under acid conditions may be involved in acid toler- involved in the glycolysis pathway and tricarboxylic acid cy- ance in L. plantarum ZDY 2013. Glutamate content was cle (Fernie et al. 2004). At pH 5.0, the increased ratio of maintained or decreased in both L. plantarum ZDY 2013 NAD /NADH in L. plantarum ZDY 2013 and L. plantarum or L. plantarum ATCC 8014 at pH 4.5, respectively. ATCC 8014 might relieve the inhibition of glyceraldehyde 3- Several previous research studies have demonstrated that 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 <0.05 676 Ann Microbiol (2017) 67:669–677 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 Acknowledgments This project was sponsored by the National Natural many amino acids may protect cells against acid stress Science Foundation of China (NSF31170091, 81760102, 31360377 and (Senouci-Rezkallah et al. 2011). Senouci-Rezkallah et al. 31260363), the Ganpo Talent Engineering 555 Project, the Academic and (2011) investigated the effects of adding glutamate and Technical Leaders Training Program for Major Subjects of Jiangxi arginine on acid tolerance of B. cereus ATCC 14579, Province (P. R. China), the Research Project of Jiangxi Provincial Education Department (GJJ13098; P. R. China), and the Postdoctoral and the results showed that cell survival at pH 4.0 in- Science Foundation Funded Project of China (M570567). creased by 1-log or 2-log populations, respectively. Deiminase and decarboxylases were mainly involved in the Compliance with ethical standards acid stress response of LAB (Fernández and Zúñiga 2006). Our data revealed that, at pH 6.2 to 5.0, ADI activity increased Conflict of interest The authors declare that they have no conflict of and then decreased gradually until pH 3.5, but it was still interest. relatively higher than the initial level. Arginine was converted Disclosures The manuscript does not contain clinical studies or patient to NH in the ADI pathway, which contributed to the data. alkalization of cell cytoplasm, protecting it from acid stress damage (Lin et al. 1995;Ryanet al. 2009). Moreover, ADI was involved in the extrusion of cytoplasmic protons of References B. cereus ATCC 14579 and regulation of intracellular pH of Lactobacillus fermentum IMDO 130101, helping cells to sur- Archibald RM (1944) Determination of citrulline and allantoin and dem- vive longer under acid stress (De Angelis et al. 2002;Senouci- onstration of citrulline in blood plasma. J Biol Chem 156:121–142 Rezkallah et al. 2011). 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A physiological comparative study of acid tolerance of Lactobacillus plantarum ZDY 2013 and L. plantarum ATCC 8014 at membrane and cytoplasm levels

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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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10.1007/s13213-017-1295-x
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Ann Microbiol (2017) 67:669–677 DOI 10.1007/s13213-017-1295-x ORIGINAL ARTICLE A physiological comparative study of acid tolerance of Lactobacillus plantarum ZDY 2013 and L. plantarum ATCC 8014 at membrane and cytoplasm levels 1 1 1 1 2 Yilin Guo & Ximei Tian & Renhui Huang & Xueying Tao & Nagendra P. Shah & 1,3 3 Hua Wei & Cuixiang Wan Received: 8 April 2017 /Accepted: 4 August 2017 /Published online: 23 August 2017 Springer-Verlag GmbH Germany and the University of Milan 2017 Abstract This study aimed to disclose the acid tolerance metabolism, increased amino acid and enzyme level) of mechanism of Lactobacillus plantarum by comparing L. plantarum ZDY 2013 can protect the cells from acid stress. L. plantarum ZDY 2013 with the type strain L. plantarum . . ATCC 8014 in terms of cell membrane, energy metabolism, Keywords Lactobacillus plantarum Acid tolerance Cell . . and amino acid metabolism. L. plantarum ZDY 2013 had a membrane Energy metabolism Amino acids 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 Introduction physiological mechanism, cell integrity was investigated through scanning electron microscopy. The study revealed Lactic acid bacteria (LAB) have been used to produce that L. plantarum ZDY 2013 maintained cell morphology fermented food over the past decades and have been de- and integrity, which is much better than L. plantarum veloped as probiotics, which are generally recognized as ATCC 8014 under acid stress. Analysis of energy metabo- safe (GRAS) (De Vries et al. 2006), for their health- lism showed that, at pH 5.0, L. plantarum ZDY 2013 en- promoting functions in the human gastrointestinal tract + + hanced the activity of Na /K -ATPase and decreased the (Duary et al. 2010). Probiotics must maintain viability in + 6 ratio of NAD /NADH in comparison with L. plantarum the gut at a high concentration (at least 10 cfu/mL) to be ATCC 8014. Similarly, amino acid metabolism of intracel- beneficial to the human host (Ferrando et al. 2015). lular arginine, glutamate, and alanine was improved in Therefore, the stronger resistance of LAB strains against L. plantarum ZDY 2013. Correspondingly, the activity of various extreme environments is a prerequisite for the de- arginine deiminase and glutamate decarboxylase of velopment of probiotic supplements. L. plantarum ZDY 2013 increased by 1.2-fold and 1.3-fold LAB are exposed to various stress conditions, such as acid, compared with L. plantarum ATCC 8014 in acid stress. In temperature, osmotic stress, or freeze drying in fermented food summary, it is demonstrated that the special physiological as well as in the gastrointestinal tract (Guchte et al. 2002). The behaviors (integrity of cell membrane, enhanced energy tolerance of LAB to these stressors is critical for screening potential candidates for LAB application. As probiotic candi- dates, bacteria have to survive under extreme acidic conditions or digestion by various enzymes in the entire gastrointestinal * Cuixiang Wan tract (Wall et al. 2007). Given that acid stress is a pivotal issue cuixiangwan@ncu.edu.cn for microbial survival, acid tolerance is generally considered as one of the criteria for selection of potential probiotics (De State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing Donglu, Nanchang, Jiangxi 330047, China Angelis and Gobbetti 2004;Parvez et al. 2006). Few publications have focused on the acid tolerance mech- Food and Nutritional Science, School of Biological Science, The University of Hong Kong, Pokfulam Road, Hong Kong, China anism of LAB such as Lactococcus lactis (Rallu et al. 2000), Lactobacillus bulgaricus (Hernandez-Hernandez et al. 2012), Jiangxi-OAI Joint Research Institute, Nanchang University, 235 Nanjing Donglu, Nanchang 330047, People’s Republic of China Lactobacillus casei (Broadbent et al. 2010), Lactobacillus 670 Ann Microbiol (2017) 67:669–677 plantarum (Hamon et al. 2014), and Lactobacillus reuteri type strain, L. plantarum ATCC 8014, were used in this study. (Teixeira et al. 2014) or on the aspect of proteomic analysis Both strains were incubated in MRS broth (Beijing Solarbio level. More routinely studied are the acid tolerance, H - Science & Technology, Beijing, China) at 37 °C for 24 h under ATPase proton pump, glutamate decarboxylase (GAD) system, anaerobic condition. Growth conditions in MRS were applied and arginine deiminase (ADI) system of bacteria involved in in all subsequent experiments. For acid tolerance analyses, the pH homeostasis in Gram-positive bacteria (Cotter and Hill pH of MRS broth was adusted to 6.2, 4.5 and 3.5. 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 analyses and acid treatment acid tolerance of Listeria monocytogenes in gastric fluid (Cotter et al. 2001); and ADI protects Bacillus cereus ATCC14579 Growth of both L. plantarum strains was assessed under dif- (Senouci-Rezkallah et al. 2011), Escherichia coli (Lin et al. ferent acid conditions (pH 6.2, 4.5 and 3.5) in MRS broth, and 1995), and L. monocytogenes (Lin et al. 1995; Ryan et al. the optical density (OD) at 600 nm was monitored at different 2009) against acid stress through the production of ammonia. time points using a microplate reader (VersaMaxTM Tunable On the other hand, alterations in cell membrane and metabolic microplate reader; Molecular Devices, Sunnyvale, CA). pathways (e.g., energy metabolic pathway and amino acid me- Additionally, the survival of L. plantarum ZDY 2013 was tabolism) also control acid tolerance (Cotter and Hill 2003). For evaluated at pH 2.5 for 1.0 h in 0.01 M PBS with L plantarum instance, an increased proportion of long-chained, monounsat- ATCC 8014 for comparison. urated fatty acids were produced in Streptococcus mutans Cells of L. plantarum ZDY 2013 and L. plantarum ATCC UA159 in response to acid stress (Fozo and Quivey 2004); 8014 at mid-exponential growth phase were centrifuged at NADH oxidase, an enzyme responsible for oxidative stress, 10,000 g for 1 min, and incubated in PBS (pH 3.5, 4.5 and and which could possibly contribute to acid resistance, its ac- 5.0) at 37 °C for 1.0 h. Then, the acid-treated and untreated tivity of L. lactis (DeFelipeet al. 1998), and Lactobacillus cells were used for further study. delbrueckii subsp. bulgaricus (Marty-Teysset et al. 2000)in- creased under acidic conditions; addition of glutamine in- creased the survival rate of L. reuteri 100–23 at pH 2.5 Scanning electron microscopy (Teixeira et al. 2014). To our best of our knowledge, few liter- ature reports have disclosed information on the physiological The morphological structure of acid treated and untreated changes of LAB due to acid stress, especially in L. plantarum. cells were examined using scanning electron microscopy Among a variety of LAB species, L. plantarum is one of (SEM). The samples were prepared as described by Bron et al. (2004), with some modifications. Briefly, cells were the mostwidelyusedstartersorprobioticsinmanyproducts (Brinques and Ayub 2011). In our previous study, a strain of harvested by centrifugation at 5000 g for10min andfixed L. plantarum ZDY 2013 was proven to be an ideal probiotic in 2.5% (v/v) glutaraldehyde for 4 h at 4 °C. Subsequently, candidate because of its tolerance to the extreme environment the cells were washed three times with deionized water, of the gastrointestinal tract. Moreover, L. plantarum ZDY then dehydrated with ethanol by using 30%, 50%, 70%, 2013 underwent a global acid tolerance reaction in terms of 80% and 90% ethanol for one time, successively, and fi- mRNA levels (Huang et al. 2015), indicating that some un- nally with absolute ethyl ethanol for three times, and then known physiological changes takes place under extreme acid- freeze-dried. The samples were fixed on an aluminum foil ic conditions. To explore the acid stress mechanisms of and sprayed with gold. 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 toler- Measurement of membrane permeability ance ability, cell integrity, and changes in energy and amino acid metabolism based on the different acid stress responses of Membrane permeability was measured using the β- L. plantarum ZDY 2013 and L. plantarum ATCC 8014. 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. Materials and methods 2014). Cells were rinsed once by centrifugation (5000 g, 5min)and resuspended in0.01MPBS(pH 7.4) toanOD Bacteria strains, media, and growth conditions 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 suspen- L. plantarum ZDY 2013, isolated from a traditional fermented sion was monitored at 420 nm using a spectrophotometer acid beans in our previous study (Huang et al. 2015), and a (Precision and Scientific, Shanghai, China). Ann Microbiol (2017) 67:669–677 671 + + Measurement of Na /K -ATPase activity cooling for 10 min, the absorbance was measured at 460 nm. The standard curve for citrulline was determined by applying + + + + The Na /K -ATPase assay was carried out using the Na /K - the same procedure to five standard solutions of citrulline (0, ATPase assay kit (Suzhou Comin Biotechnology. Suzhou, 10, 20, 30 and 40 μg/mL). One unit of activity was defined as China) following the manufacturer’s protocol. The activity the amount of enzyme catalyzing the formation of 1 μmol + + + + of the Na /K -ATPase was expressed in Na /K -ATPase cat- citrulline per hour per milligram of total protein. Protein con- alyzing ATP and the formation of 1 μmol inorganic phospho- centration in the enzyme preparation was determined as de- rus per hour per milligram of total protein. Protein concentra- scribed previously. tion was determined by the method of Coomassie Brilliant Blue staining, using bovine serum albumin as a standard Measurement of GAD activity protein. GAD activity was measured as previously described for Determination of intracellular NAD /NADH ratio L. lactis (Johnson et al. 1997; Xu et al. 2003) with minor modifications. Cells were re-suspended in 2 mL cold Cellular metabolism was stopped by putting cell suspension McIlvaine buffer (pH 4.7) combined with 0.1 mM pyridoxal into liquid nitrogen for 3 min then the content of intracellular phosphate and 1 mM mercaptoethanol, sonicated for 10 min + + NAD /NADH was determined using the NAD /NADH assay and centrifuged at 10,000 g for 10 min at 4 °C; the supernatant kit (Suzhou Comin Biotechnology) following the manufac- was taken as GAD extract. turer’s protocol. The reaction mixture consisted of enzyme extract, 10 mM L-glutamate (pH 4.8) and 0.1 mM pyridoxal phosphate. Determination of intracellular amino acids Controls without enzyme extract were included. After incuba- tion at 30 °C for 10 h, the reaction was stopped by addition of For the extraction of intracellular amino acids, 20 mL station- 200 μL 0.2 M sodium borate buffer, 1 mL 6% phenol solution ary phase cells was treated under different acid conditions and 400 μL sodium hypochloride coupled with ice water bath. (pH 6.2, 4.5 and 3.5) in 0.01 M PBS broth at 37 °C for Color development was carried out in boiling water for 1.0 h, harvested by centrifugation at 12,000 g for 10 min, 10 min, then immediately put it in an ice water bath for washed twice and resuspended in 1 mL of 0.2 M PBS, then 20 min. The optical density was read at 630 nm. The standard boiled for 15 min. Cell debris was discarded after the centri- curve for γ-amino butyric acid (GABA) was determined by fugation (12,000 g, 10 min, 4 °C). The supernatants were applying the same procedure to five standard solutions of treated at room temperature after adding 1 mL 10% TCA for GABA (0, 2, 4, 6 and 8 mM GABA). One unit of the activity 10 min. Then the mixture was centrifuged at 12,000 g for was defined as the amount of enzyme that catalyzed the for- 10 min at 4 °C, and the supernatants were analyzed with mation of 1 μmol GABA per hour per milligram of total amino-acid analyzer (Sykam, Munich, Germany). protein. Protein concentration in the enzyme preparation was determined as described previously. Measurement of ADI activity Statistical analysis ADI activity was measured as described by De Angelis et al. (2002) with some modifications. Cells resuspended in 0.05 M All results were expressed as mean ± standard deviation (SD). Tris-HCl buffer (pH 7.5) were sonicated for 10 min with an Statistical analysis was performed using two-way ANOVA ultrasonic cell disruptor (Xinzhi, Ningbo, China) and centri- procedure of SPSS 13.0 software (SPSS, Chicago, IL). Data fuged at 10,000 g for 10 min at 4 °C; the supernatant, contain- was considered statistically significant when P <0.05. ing cell wall and cytoplasm, was collected and considered as enzyme extract. The reaction mixture (2.5 mL) consisted of enzyme extract, Results 0.05 M arginine and 0.05 M acetate buffer (pH 5.5). Controls without enzyme extract were included. After incubation at Growth curve and acid tolerance ability of L. plantarum 37 °C for 1 h, the reaction was stopped by adding 500 μL ZDY 2013 and ATCC 8014 2 M HCl, and the precipitate was removed by centrifugation (10,000 g for 10 min at 4 °C). The citrulline content of the To analyze the mechanism of acid tolerance of different supernatant was determined as described by Archibald (1944) L. plantarum, the growth curve of L. plantarum ZDY 2013 1 mL supernatant was added to 1.5 mL acid mixture of and ATCC 8014 in standard or acid conditions, and their cell H PO -H SO (3:1, v/v) and 250 μL diacetyl monoxime survival in extreme acidic environment was investigated. At 3 4 2 4 (1.5%, w/v), mixed, and boiled in the dark for 30 min. After the initial pH of 6.2 (Fig. 1a), no significant difference 672 Ann Microbiol (2017) 67:669–677 between two strains was observed (P >0.05). However,the To further compare the cell integrity of both strains, cell biomass of L. plantarum ZDY 2013 was 32% higher than that membrane permeability in series acid stress was evaluated by of L. plantarum ATCC 8014 from 10 h when the initial pH using ONPG as a substrate. As shown in Fig. 3, no difference was 4.5 (Fig. 1a). The growth of L. plantarum ATCC 8014 in membrane permeability of either strain was found above was severely inhibited at pH 3.5 (Fig. 1a), whereas pH 4.5 (P > 0.05). Nevertheless, L. plantarum ZDY 2013 L. plantarum ZDY 2013 grew well and exhibited a more than presented significantly less membrane permeability at two-fold biomass increase. This finding clearly showed that pH 3.5 when compared with L. plantarum ATCC 8014 L. plantarum ZDY 2013 had superior growth performance in (P < 0.001), again indicating that L. plantarum ZDY 2013 the acidic condition. As shown in Fig. 1b, the survival rate of maintained cell morphology and integrity much better than both cells decreased in PBS at pH 2.5. When exposed to acid L. plantarum ATCC 8014 under acid stress. stress for 1 h, the viable cells reached 1.9 × 10 cfu/mL in + + L. plantarum ZDY 2013, which was more than 2 logs higher Na /K -ATPase than that of L. plantarum ATCC 8014. + + Na /K -ATPase is an integral membrane enzyme that plays a key role in maintaining electrochemical gradients. To investi- + + Differentiation of cell integrity of L. plantarum strains gate whether the Na /K -ATPase activity was related to the under acid stress acid tolerance in L. plantarum, the activity of two strains was evaluated under different acid stress (Fig. 4). No obvious dif- + + Cell morphology of L. plantarum ZDY 2013 and ATCC 8014 ference (P > 0.05) was found in the activity of Na /K -ATPase was investigated by SEM (Fig. 2). Cells grown under stan- between L. plantarum ZDY 2013 and ATCC 8014 at either dard conditions exhibited a rod-shaped, smooth-surface pH 6.2 or 4.5. However, the activity of L. plantarum ZDY morphology in both L. plantarum ZDY 2013 and ATCC 2013 and ATCC 8014 reached 8.2 ± 0.4 and 6.1 ± 0.4 U/mg, 8014 (Fig. 2a,c). When exposed to pH 3.5 for 1 h, the cells increasing by 1.88-fold and 1.62-fold at pH 5.0 when com- of L. plantarum ZDY 2013 showed a slight tendency to pared with pH 6.2. While exposed to pH 4.5, the activity was clump together, and their surfaces appeared to be less reduced to 6.4 ± 0.4 and 5.2 ± 0.4 U/mg, respectively. smooth, with some cavities (Fig. 2b). By contrast, the cells of L. plantarum ATCC 8014 showed significant changes Intracellular NAD /NADH ratio under acidic environment; many cells appeared cracked (Fig. 2d), whereas L. plantarum ZDY 2013 retained the Most energy in glucose metabolism is released by the respi- relative integrity of cell morphology at pH 3.5. ratory chain, and the physiological change that occurs during 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 OD in MRS at pH 6.2, 4.5, or 3.5. b The cell count reduction was calculated by log (N/N ); N represented the survival counts, whereas N represented the initial cell counts under acid stress of pH 2.5. All tests were performed in triplicate Ann Microbiol (2017) 67:669–677 673 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 respiration might be a consequence of the shift in the NAD / (P > 0.05), whereas at pH 3.5, arginine content increased NADH ratio (Lechardeur et al. 2011). In this study, the ratio of to 7.8 ± 0.8 and 3.9 ± 0.2 mg/mg (1.95-fold and 1.30-fold NAD /NADH in L. plantarum ZDY 2013 and ATCC 8014 over pH 6.2) in L. plantarum ZDY 2013 and ATCC 8014, under acid stress was investigated (Fig. 5). At pH 6.2, pH 4.5 respectively (Fig. 6). L. plantarum ZDY 2013 maintained and pH 3.5, no significant difference was found in the ratio of almost the same content of glutamate as in standard condi- + + NAD /NADH of the two strains (P > 0.05). At pH 5.0, NAD / tions at pH 4.5, whereas it decreased by 2.9-fold in NADH ratio in L. plantarum ZDY 2013 and L. plantarum L. plantarum ATCC 8014 (Fig. 6). A higher concentration ATCC 8014 increased to6.8 ±0.4 and10.5±1.2(P <0.01), of alanine accumulated in L. plantarum ZDY 2013 than in respectively. 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, Amino acid changes 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 To investigate the effect of amino acid metabolism on acid L. plantarum ZDY 2013 (2.6 ± 0.3 and 24.1 ± 2.8 mg/mg, tolerance mechanisms of L. plantarum, the chang in intra- respectively) but not in L. plantarum ATCC 8014 during acid cellular amino acid content of the two strains was moni- stress. These results suggested that the accumulation of argi- tored. Under standard conditions, no significant difference nine, glutamate, and alanine might contribute to the acid toler- was found in arginine, glutamate, and alanine content ance response in L. plantarum ZDY 2013. 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 <0.05 674 Ann Microbiol (2017) 67:669–677 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 ADI activity probiotic supplements. In our previous study, L. plantarum ZDY 2013 was found to initiate a complex metabolic network The effect of pH on ADI activity was further determined in involving cell membrane components, cellular metabolism, both strains. As shown in Fig. 7, at pH 5.0, ADI activity in- and energy production under acid stress, as reported at the creasedto30.0 ± 1.8and 25.09 ± 1.8U/mgin L. plantarum molecular level by Huang et al. (2015). However, the related ZDY 2013 and ATCC 8014, respectively. The activity de- physiological mechanisms of the acid tolerance response are creased at pH below 5.0 in both strains, but no significant yet to be clarified. In this study, we performed a comparative difference was found between them (P > 0.05) at pH 6.2. and systematic study of two L. plantarum strains, namely, This result demonstrated that ADI was involved in the acid L. plantarum ZDY 2013 and L. plantarum ATCC 8014, and tolerance mechanism response in L. plantarum ZDY 2013. disclosed their acid tolerance response with regard to the cell membrane, energy metabolism, and amino acid metabolism. GAD activity Generally, the fluidity and integrity of the bacterial cyto- plasmic membrane affects the viability of cells and their met- Acid stress might affect the GAD activity of cells of abolic functions, particularly under stress conditions L. plantarum ZDY 2013 and ATCC 8014. As shown in (Mykytczuk et al. 2007), since the cell envelope plays an Fig. 8, at pH 4.5, GAD activity of L. plantarum ZDY 2013 important role in cellular growth, host defense, and in main- and ATCC 8014 increased by 3.3-fold and 2.7-fold, respec- taining the stability of the intracellular environment (Wu et al. tively, compared with that at pH 6.2. Although GAD activity 2012). In our study, L. plantarum ZDY 2013 retained the increased in the two strains at pH 5.0, no significant differ- relative integrity of cell morphology and the structure of in- ence was found between them (P > 0.05) at pH 6.2. tracellular 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 Discussion 2013 in acid stress; for instance, the survival rate of L. plantarum ZDY 2013 was approximately 100-fold higher L. plantarum has a great potential to survive in acid stress than that of L. plantarum ATCC 8014 in pH 2.5 after 1 h. environments either in vitro or in vivo; therefore, the acid Similar to many other microorganisms under acid stress, tolerance properties of L. plantarum have attracted much at- L. plantarum might start pH homeostasis activities including tention because of its broad application in fermented food and H -ATPase proton pump, alterations in cell membrane, and 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 Ann Microbiol (2017) 67:669–677 675 Fig. 6 Changes in intracellular arginine, glutamate, and alanine determined. The unit of mg/mg indicates the amount of amino acids concentration L. plantarum ZDY 2013 and L. plantarum ATCC 8014. (mg) per milligram of protein. All tests were performed in triplicate. Concentration of intracellular arginine, glutamate, and alanine was Statistically significant difference was at P <0.05 metabolic pathways (Cotter and Hill 2003) to enhance its sur- phosphate dehydrogenase activity because a higher intracellu- + + + vival efficiency. Na /K -ATPase is a ubiquitous integral mem- lar NAD /NADH ratio could affect the enzymes using NAD brane enzyme (Towle 1984), which maintains the ion gradi- or pyruvate as substrate (Lechardeur et al. 2011; Wendisch + + + ents of Na and K by consuming energy through ATP hydro- et al. 2006). An increased intracellular NAD /NADH ratio lysis (Jutfelt 2006). In our study, we confirmed that and decreased levels of the content intracellular NAD and L. plantarum ZDY 2013 strengthened proton cell pumping NADH could contribute to the increased of ATP/ADP ratio + + as supported by a 1.88-fold increase in Na /K -ATPase activ- and enhance the energy metabolism (Lin et al. 2009). ities from an initial level that was significantly higher than that Amino acid metabolism plays an important role in of L. plantarum ATCC 8014 in the same pH of 5.0 (P <0.05). maintaining the homeostasis of intracellular pH, generat- A similar result was reported also in L. reuteri (Lee et al. ing metabolic energy, and enhancing the cell resistance to + + 2008). The activity of Na /K -ATPase decreased at pH 4.5. environmental stress of LAB (Wu et al. 2013). In our Wang et al. (2002) revealed that the active transport of Na work, we demonstrated that the arginine and alanine con- and K was inhibited at pH 4.6–5.0 in their experiment. In our tent of cells increased significantly for L. plantarum ZDY previous work, the gene transcription levels in ATP synthase 2013 (P < 0.001), but was maintained or reduced for (atpA and atpC) and glycerol-3-phosphate dehydrogenase L. plantarum ATCC 8014 at pH 3.5. L. plantarum ZDY (gspA) related to cellular energy metabolism were enhanced 2013 shifted the metabolic pathway by increasing the flux during acid stress (Huang et al. 2015). to arginine under acid conditions, and, based on arginine Normally, NAD(H) plays an important role in cell death deiminase system, higher intracellular arginine concentra- (Ying 2008), and can affect numerous enzymatic activities tion under acid conditions may be involved in acid toler- involved in the glycolysis pathway and tricarboxylic acid cy- ance in L. plantarum ZDY 2013. Glutamate content was cle (Fernie et al. 2004). At pH 5.0, the increased ratio of maintained or decreased in both L. plantarum ZDY 2013 NAD /NADH in L. plantarum ZDY 2013 and L. plantarum or L. plantarum ATCC 8014 at pH 4.5, respectively. ATCC 8014 might relieve the inhibition of glyceraldehyde 3- Several previous research studies have demonstrated that 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 <0.05 676 Ann Microbiol (2017) 67:669–677 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 Acknowledgments This project was sponsored by the National Natural many amino acids may protect cells against acid stress Science Foundation of China (NSF31170091, 81760102, 31360377 and (Senouci-Rezkallah et al. 2011). Senouci-Rezkallah et al. 31260363), the Ganpo Talent Engineering 555 Project, the Academic and (2011) investigated the effects of adding glutamate and Technical Leaders Training Program for Major Subjects of Jiangxi arginine on acid tolerance of B. cereus ATCC 14579, Province (P. R. China), the Research Project of Jiangxi Provincial Education Department (GJJ13098; P. R. China), and the Postdoctoral and the results showed that cell survival at pH 4.0 in- Science Foundation Funded Project of China (M570567). creased by 1-log or 2-log populations, respectively. Deiminase and decarboxylases were mainly involved in the Compliance with ethical standards acid stress response of LAB (Fernández and Zúñiga 2006). Our data revealed that, at pH 6.2 to 5.0, ADI activity increased Conflict of interest The authors declare that they have no conflict of and then decreased gradually until pH 3.5, but it was still interest. relatively higher than the initial level. Arginine was converted Disclosures The manuscript does not contain clinical studies or patient to NH in the ADI pathway, which contributed to the data. alkalization of cell cytoplasm, protecting it from acid stress damage (Lin et al. 1995;Ryanet al. 2009). Moreover, ADI was involved in the extrusion of cytoplasmic protons of References B. cereus ATCC 14579 and regulation of intracellular pH of Lactobacillus fermentum IMDO 130101, helping cells to sur- Archibald RM (1944) Determination of citrulline and allantoin and dem- vive longer under acid stress (De Angelis et al. 2002;Senouci- onstration of citrulline in blood plasma. J Biol Chem 156:121–142 Rezkallah et al. 2011). 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Annals of Microbiology – Springer Journals

Published: Aug 23, 2017

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