Response of Lactobacillus plantarum VAL6 to challenges of pH and sodium chloride stresses

To investigate the effect of environmental stresses on the exopolysaccharide biosynthesis, after 24 h of culture at 37 °C with pH 6.8 and without sodium chloride, Lactobacillus plantarum VAL6 was exposed to different stress conditions, including pH (pHs of 3 and 8) and high sodium chloride concentration treatments. The results found that Lactobacillus plantarum VAL6 exposed to stress at pH 3 for 3 h gives the highest exopolysaccharide yield (50.44 g/L) which is 6.4 fold higher than non-stress. Under pH and sodium chloride stresses, the mannose content in exopolysaccharides decreased while the glucose increased in comparison with non-stress condition. The galactose content was highest under stress condition of pH 8 meantime rhamnose content increased sharply when Lactobacillus plantarum VAL6 was stressed at pH 3. The arabinose content in exopolysaccharides was not detected under non-stress condition but it was recorded in great amounts after 3 h of stress at pH 3. In addition, stress of pH 8 triggered the mRNA expression of epsF gene resulting in galactose-rich EPS synthesis. According to our results, the stresses of pH and sodium chloride enhance the production and change the mRNA expression of epsF gene, leading to differences in the monosaccharide composition of exopolysaccharides.

Exopolysaccharides (EPS) are high molecular weight biological polymers synthesized extracellularly by various microorganisms (archaea, bacteria, fungi or algae) and applied in the variety of industries such as food, pharmaceuticals, etc. 1 . Especially, Lactic acid bacterial (LAB) are the most remarkable group of EPS producing bacteria because of their strong production of EPS 2 and they are GRAS (Generally Recognized As Safe) 3 . Dextran, levan, oligosaccharides, etc. are polysaccharides produced by LAB and widely used in the food industry to improve the rheological, structural and sensory properties of fermented products 2 . In addition, LAB's EPS can also possess biological functions such as immune stimulation, antitumour effects, prebiotic activities and antioxidants 4 .
The function of EPS for bacteria is to protect cells from the negative effects of environment as dehydration, antibiotics, phagocytosis and phage attacks because of their role in forming biofilms [5][6][7] . Changes in environmental conditions may alter the EPS production 8 and result in the biosynthesis of new types of EPS in bacteria 9 . The final EPS yield and the characteristics of EPS are strongly influenced by environmental conditions 10,11 . Fedorová et al. 12 have found a positive correlation between EPS production and L. reuteri resistance to low pH stress. Seesuriyachan et al. 13 also reported that EPS production increased when L. confusus was stressed under high salinity. Moreover, the effect of various environmental conditions can trigger the expression of genes involved in the biosynthesis of EPS in LAB. For example, when the pH of the culture medium decreased from pH 6.5 to pH 5.5, the eps gene cluster expression increased in S. thermophilus ASCC 1275 14 .
The EPS biological properties are determined by its monosacharide composition. The differences in the ratio and composition of monosaccharides cause the alteration of biological activity and therapeutic effect 15 . For instance, the anti-inflammatory activity of EPS is related to the sugar ratio of galactose, rhamnose and glucose. Galactose-rich EPS enhances their anti-inflammatory activity 16 . Rare sugar-rich EPS have a higher biological activity than EPS consisting of common sugars. Some of rare sugars as L-fucose, L-rhamnose, and uronic acid contain valuable properties which maybe attractive to a large number of applications for various fields including antioxidant, anti-inflammatory substances, etc.; particularly, such rare sugars can be used to synthesize the nucleoside substances which have antiviral effects 17 . Rhamnose-rich EPS secreted by L. paracasei have ability to boost the immune system 18 23 . Although there are many reports on the relationship between changed environmental conditions and EPS biosynthesis, studies on the effects of environmental stress on EPS synthesis in Lactobacilli are still limited. L. plantarum has been known to be a producer of EPS with distinct biological properties and activities for different applications, including health and food industry 24 . Thus, this study focuses on assessing the impact of stress conditions such as pH (low and high) and high sodium chloride concentration on EPS synthesis in L. plantarum VAL6. The purpose of this work is to improve the yield and monosaccharide composition of EPS for highly bioactive EPS production.

Results
Effect of pH and sodium chloride stresses on EPS production. The harsh environmental conditions stimulate microorganisms to develop various adaptive strategies, allowing them to cope with the adverse effects of extreme conditions. Among these strategies, EPS biosynthesis is one of the most common protection mechanisms 11 . We wonder if it is true during L. plantarum VAL6′s EPS production under pH and sodium chloride stresses. Therefore, L. plantarum VAL6 was treated with stress conditions of pH 3, pH 8 and high concentration of sodium chloride. As results of our data, EPS yield increases considerably under stress treatments of pH 3 and pH 8 compared to non-stressed condition (Fig. 1a).
The amount of EPS produced by L. plantarum VAL6 under stress of pH 3 was higher than that under other stress conditions. The highest EPS yield produced by ones was over 50 g/L after exposure to stress at pH 3 for 3 h and 6.4 fold higher than the control (only 7.8 g/L). While under stress at pH 8, the yield of EPS was about 16 g/L and 2.1 fold higher. These results indicated that L. plantarum VAL6 enhances EPS synthesis under stress conditions of pH 3 and pH 8.
The production of EPS was influenced by not only pH stress but also sodium chloride stress. The effect of sodium chloride stress on EPS production in L. plantarum VAL6 was determined in the presence of sodium chloride (10%). It is observed that there is a slight increase in the production of EPS compared to non-stress (Fig. 1a). Under sodium chloride stress, the yield of EPS fluctuated approxymately 10 g/L. However, there was still a significant difference (P < 0.05) in EPS yield between sodium chloride stress and non-stress.
In addition to the evaluation of EPS production, the growth capacity of L. plantarum VAL6 under stress conditions was also examined. The results showed that L. plantarum VAL6 is able to survive during the time of tested stresses (Fig. 1b). Under the stress conditions of NaCl and pH 8, the density of L. plantarum VAL6 was found an insignificant difference (P > 0.05) compared to non-stress. However, the density of L. plantarum VAL6 decreased strongly under stress condition of pH 3.
The changes in the monosaccharide compositions of EPS. Environmental conditions greatly affect the yield, monosaccharide composition as well as functional properties of EPS synthesized by bacteria 10,11 . To better clarify the effect of environmental conditions on EPS biosynthesis, we have evaluated the impact of pH and sodium chloride stress conditions on changes in the monosaccharide composition of EPS. The results were shown in Fig. 2.
The ratio of monosaccharides in EPS differently fluctuated between non-stress and stress conditions. Stress conditions decreased the ratio of mannose in EPS (Fig. 2a). Under stress of sodium chloride, the percentage of mannose fluctuated around 81% during the time of stress and was lower than non-stress samples (84.33%). For stress of pH 3, the percentage of mannose was 83.46% after 1 h of stress but then dropped to 75-76% when extending the stress period. For stress of pH 8, the proportion of mannose in EPS was only 73-74%. www.nature.com/scientificreports/ In contrast, stress conditions increased glucose levels (Fig. 2b). Under stress of pH 3, the glucose content in EPS increased from 12.55% after 1 h of stress to a maximum ratio of 15.77% after 3 h and then it steadily decreased to 14.21% after 7 h of stress. Whereas under stress of pH 8, the glucose ratio fluctuated between 17.15 and 18.59%. The glucose content in EPS under sodium chloride stress increased slightly from 13.88% after 1 h of stress to a approximate maximum of 14.7% at 3 h and then it steadily decreased according to the time of stress. These compared with about 13% under non-stress condition. www.nature.com/scientificreports/ The galactose ratio in EPS produced by L. plantarum VAL6 in exposure to stress at pH 8 sharply increased (Fig. 2c). Under stress of pH 8, the galactose ratio ranged 6.20-6.49% compared to 1.21% of the control. This value was only about 0.62-1.75% under stress of pH 3. It was also observed that galactose ratio in EPS slightly increased when L. plantarum VAL6 was exposed to sodium chloride stress and ranged 2.7-3%.
Research results also indicated that there was a difference in the rhamnose content of EPS when L. plantarum VAL6 was exposed to pH stress conditions (Fig. 2e). Especially, the rhamnose content of EPS from L. plantarum VAL6 stressed at pH 3 was highest and increased gradually following to the time of stress. It was approximately 5% after 7 h of stress, 6.6 times higher than non-stress. Under stress of pH 8, the ratio of rhamnose fluctuated approximately 2% and it was did not detected during stress periods over 3 h. For sodium chloride stress, the ratio of rhamnose was about 1%.
Arabinose was not detected in EPS composition under non-stress condition, but it was found under tested stress conditions (Fig. 2d). Under stress of pH 3, the arabinose content in EPS was considerably higher than that under stress conditions of pH 8 and sodium chloride. The arabinose ratio was highest when L. plantarum VAL6 exposed to stress at pH 3 for 3 h. It peaked at 4.25% compared to just about 1% under stress conditions of pH 8 and sodium chloride. In contrast, xylose was found in the EPS composition of the controls although it was quite small. However, it was not detected in EPS produced by L. plantarum VAL6 under tested stress conditions (Fig. 2f).

Expression of EPS genes. Changes in expression of genes involved in EPS biosynthesis under different
environmental conditions have been previously reported 25,26 . Assuming that alterations in the monosaccharide composition of EPS produced by L. plantarum VAL6 under pH and sodium chloride stresses could be also related to the expression of eps genes, we analyzed the expression of the two genes including epsD (encoding a putative protein tyrosine phosphatase) and epsF (encoding a putative glycosyltransferase) 27 (see "Materials and methods" section for the primers used to amlify cDNA, Table 2). Results showed that epsD was expressed both stress and non-stress conditions (Fig. 3). Meanwhile, epsF was only expressed under stress of pH 8 (Fig. 3).

Discussion
The results showed that stress of pH 3 stimulates L. plantarum VAL6 to strongly synthesize EPS. It is explained that EPS affect cell's sensitivity to acid pH. Anions linked to EPS can limit acid's access to bacterial cells 28 . Moreover, cultures were stressed at low pH by addition of H 3 PO 4 . Therefore, having been synthesized, EPS may be attached to phosphate groups from H 3 PO 4 by phosphorylation in order to form phosphate polysaccharide leading to increase the weight of obtained EPS. The presence of phosphate groups in EPS has been also recorded in some studies. EPS-b and EPS-r produced by L. plantarum EP56 contained phosphate groups which contribute to their negative charge 29 . Phosphate groups may provide important properties to EPS because they are required for the activation of lymphocytes 30 . The negative charge on EPS surface plays an important role in isolating positively charged toxic compounds such as nisin and metal ions 31 . In this study, phosphate rich EPS may be synthesized by L. plantarum VAL6 under stress condition of pH 3. Several studies also demonstrated that there is a correlation between low pH and EPS production. The contemporary pH decrease and the presence of sugar (osmotic stress) stimulated EPS production in LAB 32 . The EPS producing L. mucosae DPC 6426 exhibited a fivefold increasing survival during a 60-min exposure to HCl compared to EPS non-producing L. mucosae DPC 6420 21 . The strains of L. reuteri producing more EPS showed a considerably higher tolerance at low pH. The resistance of L. reuteri to the stress conditions of gastrointestinal tract was related to EPS production 12 . For treatment of pH 8, although EPS yield was lower compared to treatment pH 3, it was still higher than the control sample. This result indicated that L. plantarum VAL6 also enhances EPS synthesis to cope with the effects of high pH. There are few studies reporting on relationships between alkaline pH and EPS production. However, it was found that EPS related to flocs formation, composed of a complex mixture of EPS, provide microorganism protection against alkaline pH 33 . www.nature.com/scientificreports/ The high concentration of sodium chloride affected positively EPS production in L. plantarum VAL6. The increase in EPS production was related to sodium chloride tolerance 34 . The extracellular polysaccharide layer with high water retention helps microorganisms for better resistance to osmosis 35 . Another study has also demonstrated that L. confusus TISTR 1498 overproduced EPS under high salinity stress 13 . The effect of sodium chloride on the biosynthesis of EPS in LAB has been little reported. However, many studies on cyanobacteria demonstrated that high sodium chloride concentration positively affects EPS production. There was a considerable increase in EPS production of Synechocystis sp. exposed to sodium chloride 34 . The production of extracellular carbohydrates by Microcoleus vaginatus also considerably increased under higher salt concentrations 36 .
The results of growth kinetics demonstrated that L. plantarum VAL6 is able to survive during tested stresses. Despite a sharp decrease, the density of L. plantarum VAL6 was still higher than 5 log CFU/mL after 7 h of stress at pH 3. Many studies have also demonstrated that L. plantarum can outlive under extremely low pH condition. According to the study of Lee et al. 37 , some L. plantarum strains isolated from Korean kimchi could survive at pH 2.5 for 2 h. The density of L. plantarum ZDY 2013 altered inconsiderably at pH 3 and could survive for 6 h at pH 2 at over 7 log CFU/mL 38 .
This research characterised monosaccharides making of EPS produced by L. plantarum VAL6 with and without exposure to pH and sodium chloride stresses, then quantified the monosaccharide compositions by GC-FID. Overall, monosaccharide composition of EPS produced by L. plantarum VAL6 was composed of different monomers. The presence of mannose, glucose and galactose was common in both non-stress and stress conditions. The EPS of L. plantarum VAL6 from non-stress samples was found to be comprised of five identifiable monosachharides. In descending order, mannose, glucose, galactose were three major monosaccharides present in these samples. This result was not the same as other L. plantarum species studied previously such as L. plantarum EP56 29 , L. plantarum C88 39 , L. plantarum YW11 40 , where glucose was main component sugar. However, the monosaccharide ratio of EPS produced by L. plantarum VAL6 was similar to the monosaccharide ratio of EPS produced by L. plantarum 70810 (18.21 Glc: 3.03 Gal: 78.76 Man) 41 . Likewise, mannose, glucose and galactose (in a molar ratio of 12.94:7.26:3.31) were the main composition of EPS produced by L. plantarum KX041 15 .
The monosaccharide composition of EPS from L. plantarum VAL6 exposed to tested stress treatments changed both monosaccharide type and the ratio of sugars. This result suggested that stresses of pH and sodium chloride can trigger the reprogramming of cellular mechanism for EPS biosynthesis resulting in changes in monosacchride composition of EPS with the accumulation of certain common and rare sugar ratios (Table 1). Remarkably, the result found a substantial increase in rhamnose content under stress condition of pH 3. Rhamnose-rich EPS show interesting biological activities which make them likely to be used for many applications such as pharmaceuticals, cosmetics and functional foods 42,43 . For instance, rhamnose and fucose have gotten more attention when oligo and polysaccharides rich in rhamnose and fucose were found to be associated with anti-aging skin mechanisms 44 . Also according to this study, galactose content in EPS increased sharply when L. plantarum VAL6 was exposed to stress of pH 8. As reported by previous studies, galatose-rich EPS could enhance anti-inflammatory activity 16 . Galactose-rich EPS also increased the adhesion and capacity of biofilm formation in LAB 45 . Another interesting result found in our study was the presence of arabinose in EPS under tested stresses but it was not detected under non-stress condition. This result suggested that arabinose component in EPS may be involved in bacterial stress resistance. A prior study has also demonstrated that the arabinose component in EPS plays an important role in cell aggregation to deal with hostile environmental conditions 46 . These results elicit the using potential of pH stress for the production of carbohydrate bioactive compounds.
The changes in the monosaccharide composition of EPS were related to the expression of tested eps genes. epsD gene takes part in the functional group which encodes enzymes involved in phosphoregulatory system, it controls polysaccharide assembly 20 . This may explain why epsD is always expressed in EPS synthesis. The results of this study also showed that epsD was expressed under non-stress and tested stress conditions (Fig. 3). However, epsF gene was only expressed under stress condition of pH 8 (Fig. 3). Furthermore, the result of study also demonstrated that stress of pH 8 strongly increased galactose content in EPS (Fig. 2c). In S. thermophilus Sfi6, epsF has been shown to encode a galactosyltransferase 47 , a transport enzyme which adds galactose to the repeating unit of EPS structure, leading to increase galactose proportion in the monosaccharide composition of EPS. Similarly, the results of this study suggested that the expression of epsF gene is related to the synthesis of galactose-rich EPS in L. plantarum VAL6.  www.nature.com/scientificreports/ Conclusion L. plantarum VAL6 was able to survive and enhance EPS production under pH and sodium chloride stresses. The highest yield of EPS reached to 50.44 g/L when L. plantarum VAL6 was exposed to stress of pH 3. The results also demonstrated that the stresses of pH 8 and sodium chloride positively affect the EPS producing ability of L. plantarum VAL6. Futhermore, the monosaccharide compositions in EPS synthesized by L. plantarum VAL6 differently changed under the impact of these stresses. Under tested stress conditions, the content of mannose decreased while glucose content increased compared to non-stress circumstance. Galactose content dramatically increased when L. plantarum VAL6 was exposed to stress of pH 8. There was an increase in the proportion of rare sugars (rhamnose and arabinose) when L. plantarum VAL6 was exposed to stress treatments. Rhamnose content in EPS was highest when L. plantarum VAL6 was stressed at pH 3 for 7 h. A large amount of arabinose was found in EPS when L. plantarum VAL6 was exposed to stress at pH 3 for 3 to 7 h. There was a relationship between epsF gene expression and high galactose content in EPS synthesized under stress of pH 8. These results elicit the biosynthesis of EPS in order to enrich rare sugars by using environmental stresses. The EPS with higher proportion of rare monosaccharides which is capable of increasing their biological properties can be used for different applications.

Materials and methods
Microbial strain, cultivation medium and stress conditions. Lactobacillus plantarum VAL6 (or Lactiplantibacillus plantarum VAL6, according to the taxonomy of the new genus Lactobacillus described by Zheng et al. 48 ) was isolated from pickled vegetables. The strain was identified by molecular method using 16S rRNA sequence analysis and amplification to check the recA gene fragment. The primers used for amplifying the 16S rRNA region were 27f. (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492r (5′-GGT TAC CTT GTT ACG ACT T-3′) as forward and reverse primers, respectively 49 . The primers used for recA gene fragment amplification were planF (5′-CCG TTT ATG CGG AAC ACC TA-3′), and pREV (5′-TCG GGA TTA CCA AAC ATC AC-3′) 50 .
The amplification of recA gene region Cultures were performed on Man-Rogosa-Sharpe medium (MRS) 51 . All the cultures have been carried out in a 5 L bioreactor (BIOSTAT, Sartorius Stedim Biotech GmbH, Germany). Briefly, 5 L of MRS medium was inoculated with 100 mL of overnight bacterial culture (OD 595 = 1.5). pH was maintained to 6.8 (regulation by addition of NaOH 10 M), temperature was kept at 37 °C, agitation rate was set up to 250 rpm under aerobic facultative condition. For pH stress, after 24 h of culture at 37 °C and pH 6.8, the culture was treated with pH conditions either pH 3 or pH 8 for 7 h. The time was calculated when the bioreactor reached the required pH. For sodium chloride stress, after 24 h of culture at 37 °C and pH 6.8, the culture was treated by adding NaCl to reach a concentration of 10% for 7 h. The non-stress control was simultaneously carried out in another bioreactor with pH 6.8 for the entire time. Under both stress and non-stress conditions, temperature was maintained at 37 °C and agitation rate was set up to 250 rpm. A volume of 500 mL of culture was sampled in every hour during the stress treatment for EPS quantification and characterization.
EPS extraction and quantification. EPS was extracted from cell suspensions collected at the given time point according to the method described by Salazar et al. 52 and Nguyen et al. 53 . Crude EPS total produced by L. plantarum VAL6 was harvested after 24 h by mixing 100 mL of supernatant with an equal amount of NaOH 2 M and was gently stirred overnight at room temperature. Supernatants were then recovered by centrifugation at 8400 g for 20 min and crude EPS was precipitated from the supernatants by adding twice the volume of 96% (v/v) cold ethanol. The precipitation was carried out at 4 °C for 48 h. After the second centrifugation step at 8400 g and 4 °C for 30 min, EPS was dried at 55 °C until constant weight. Consequently, the total EPS yield was determined gravimetrically by calculating the total weight of EPS per liter of culture medium.

Compositional analysis of EPS by GC-FID. Monosaccharide composition of EPS was analyzed by GC-
FID according to the method described by Yuan et al. 54 with some modify 54 . 0.1 g of EPS was hydrolyzed with 3 mL of 2 M trifluoroacetic acid (TFA) at 105 °C for 4 h. The hydrolysed sugars were derivatised by the addition of hydroxylamine, pyridine and were subjected to acetylation using acetic anhydride (CH 3 CO) 2 O. The derivative products were used for determination of the monosaccharide composition by Gas Chromatography (GC). Sample of 1 μL was injected into the GC-FID using autosampler. GC was performed on Agilent 6890 N (USA) and HP-5MS UI column (30 m length 0.25 mm inner diameter 0.25 μm film thickness). Nitrogen was used as the carrier gas with 1 mL/min flow rate. The chromatographic conditions used are as follows: the initial column temperature was held at 120 °C for 2 min, increased at a rate of 20 °C/min to 200 °C for 4 min, and then subsequently increased at 25 °C/min to 280 °C, where it was held at 290 °C for 5 min. The comparison was made with standard mannose, glucose, galactose, rhamnose, arabinose, xylose and fucose for sugar identification. Examine the expression of eps genes. Total RNA was extracted from 500 μL cell suspension of L. plantarum VAL6 using TRIzol reagent (Invitrogen, UK) according to the manufacturer's instruction. RNA was treated with RQ1 RNase-free DNase (Promega, Madison, USA) to remove contamination of chromosomal DNA. Qualitative test of RNA at 260 and 280 nm was found to be. more than 1.8. One-Step RT-PCR was performed to synthesize cDNA from 1 μg of the DNase-treated RNA using HiSenScript RH(-) cDNA Synthesis Kit (iNtRON, Korea). Conventional PCR was done to amplify complementary DNA (cDNA). The reaction mixture (48 µL of master mix: 5 µL of 10X buffer, 0.5 µL of 10 mM dNTP mixture, 2 µL of 5 μM primers, 0.5 µL of Taq and 40 µL of distilled water) was mixed with 2 µL of template cDNA. The primers were shown in Table 2. The cycle conditions were as follows: 95 °C for 3 min; 30 cycles of 95 °C