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Purification and characterization of a novel exopolysaccharides produced by Lactobacillus sp. Ca6
International Journal of Biological Macromolecules 74 (2015) 541–546
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Purification and characterization of a novel exopolysaccharides produced by Lactobacillus sp. Ca6 Imen Trabelsi, Sirine Ben Slima, Hela Chaabane, Ben Salah Riadh ∗ Laboratory of Microorganisms and Biomolecules (LMB), Centre of Biotechnology of Sfax, University of Sfax, Road of Sidi Mansour Km 6, P.O. Box 1177, Sfax 3018, Tunisia
a r t i c l e
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Article history: Received 1 December 2014 Received in revised form 16 December 2014 Accepted 19 December 2014 Available online 15 January 2015 Keywords: Exopolysaccharide (EPS) FTIR spectroscopy Probiotics
a b s t r a c t This study was undertaken to investigate the ability of ten lactic acid bacterial strains to produce exopolysaccharides (EPS) on MRS broth containing 4% sucrose. A maximum EPS production yield of 2.4 g/l was obtained by strain Lactobacillus sp. Ca6 . The results from thin layer chromatography (TLC) and high performance chromatography (HPLC) analyses showed that the EPS produced was a polymer of glucose. Further FTIR spectroscopic analysis revealed the presence of carboxyl, hydroxyl and amide groups corresponding to a typical EPS. In addition to EPS production, Lactobacillus sp. Ca6 displayed good probiotic properties (antimicrobial activities and sensitivity to several antibiotics) and resistance to acidic condition (pH 2) and 5% bile bovine. Overall, the findings indicate that this strain has a number of promising properties that make it a potential promising candidate for future application as a food additive. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The increased demand of natural polymers for various industrial applications in recent years has led to a renewed interest in the production of exopolysaccharides (EPS) from natural origins [1]. Several microorganisms, including lactic acid bacteria (LAB), fungi and plants, have an ability to synthesize extracellular polysaccharides as soluble or insoluble polymers [2–4]. Several EPS produced by natural microorganisms have been employed in the food, cosmetic, and pharmaceutical industries where they serve various functions, such as bioflocculants, bioabsorbents, heavy metal removal agents, and drug delivery agents [5]. Exopolysaccharides from microbial origins include dextrans, xanthan, gellan, pullulan, yeast glucans and bacterial alginates [4,6]. Various microbe-derived polysaccharides have been used as food additives, including xanthan from Xanthomonas campestris [7–9] and gellan from Pseudomonas elodea [10]. Several LABs have gained increasing attention in recent research particularly because of the textural properties they can impart to several biotechnological products and the “Qualified Presumption of Safety” (QPS) status they have acquired [11]. In fact, the implementation of functional cultures that “contribute to food safety and/or offer one or more organoleptic, technological and
∗ Corresponding author. Tel.: +216 74 87 04 51; fax: +216 74 87 04 51. E-mail addresses: riadh [email protected], [email protected] (B.S. Riadh). http://dx.doi.org/10.1016/j.ijbiomac.2014.12.045 0141-8130/© 2015 Elsevier B.V. All rights reserved.
nutritional, or health advantages” in the manufacture of fermented foods has gained growing attention in the last few decades [12,13]. In this context, several LABs, including Lactobacillus reuteri, have been reported for their ability to produce EPS for application as safe and healthy alternative bio-thickeners in various food products [14]. Based on their composition and biosynthesis mechanisms, EPS have been divided into two classes: heteropolysaccharides and homopolysaccharides. Heteropolysaccharides consist of multiple sugar types and are synthesized by the combined action of different types of glycosyltransferase enzymes [15]. Homopolysaccharides are generally synthesized from the sole sucrose substrate by the action of the sucrase enzyme [16]. Sucrase synthesizes polysaccharides consisting of either glucose (glucan) or fructose (fructan) residues [16]. In general, glucans and fructans can be used as viscosifying, stabilizing, emulsifying, sweetening, gelling, or waterbinding agents in the food, as well as in non-food, industry [17,18]. Sucrase genes of Leuconostoc, Streptococcus and L. reuteri strains, including L. reuteri ATCC 55730, have been characterized [19–23]. There is strong evidence in the literature that EPS have good emulsifying activity, a property that is highly desired for various food formulations [24,25]. EPS have also been shown to have important therapeutic activities [18,26,27]. The viscosity and biological activities of EPS depend on their primary structure, molecular mass, and sugar composition [28]. The amount of EPS production depends on the medium and cultural conditions used for microbial growth. The present study focused on
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the screening of ten lactic acid bacteria for their ability to produce EPS in sucrose-MRS broth. The EPS was separated, purified, and characterized by TLC, HPLC and FTIR-spectroscopy. The selected strain and its derivatives were evaluated in vitro as potential probiotics, including their resistance to acidic pH, bile bovine and antibiotics and their ability to inhibit pathogenic bacteria. 2. Materials and methods 2.1. Screening of produced EPS LABS from sucrose Ten LAB strains, which had previously been isolated in our laboratory from the gastrointestinal tract of indigenous poultry in Tunisia [29], were screened for their ability to produce EPS from sucrose. The samples were plated on De Man, Rogosa and Sharpe (MRS) agar media containing 4% (w/v) sucrose, 10 g/l tryptone, 10 g/l meat extract, 5 g/l yeast extract, 5 g/l sodium acetate, 2 g/l disodium phosphate, 2 g/l tri-ammonium citrate, 0.1 g/l MgSO4 , and 0.05 g/l MnSO4 (pH 6.5). After incubation for 24–48 at 30 ◦ C under anaerobic conditions, the plates were checked for the presence of colonies displaying a mucoïd aspect. 2.2. EPS extraction and purification The EPS was purified by a slightly modified method described by Garcîagaribay and Marshall [7]. In brief, strains Ca6 and TN9 were grown in 100 ml MRS-sucrose in an Erlenmeyer flask at 30 ◦ C for 48 h in anaerobic jars containing GasPak (Oxoid). The flasks were then taken out and heated at 100 ◦ C for 30 min to dissolve cell attached EPS and centrifuged at 12,000 rpm for 15 min. Crude EPS was precipitated by the addition of 3 volumes of chilled ethanol (95%) to the supernatant. After precipitation at 4 ◦ C (overnight) the sample was centrifuged at 12,000 rpm for 15 min, and the pellet was retained. The sample was redissolved in distilled water and lyophilized. The polymer dry mass of purified EPS was determined by measuring the mass of the precipitate. 2.3. Monosaccharide composition of EPS The purified EPS (2 mg) was hydrolyzed in 250 l of 2 M trifluoroacetic acid (TFA) at 100 ◦ C for 1 h. Excess TFA was removed by evaporation, and the hydrolysate was washed thoroughly with water and lyophilized. The lyophilized powder was dissolved in 100 l of water, and a 5-l aliquot was used for thin-layer chromatography (TLC). Migration was performed twice on a silica gel TLC plate (20 cm × 20 cm) using n-butanol:ethanol: water (2:1:1, v/v). Carbohydrates were visualized by heating the TLC plate after spraying with 5% (v/v) sulfuric acid in ethanol. Glucose, fructose, sucrose and xylose were used as standard monosaccharides. The supernatant was then used for the analysis of monosaccharides in the hydrolysate. A 20-l sample of the supernatant was added to 980 l of H2 SO4 and filtered through a 0.45 m pore size filter. Monosaccharide composition was analyzed by HPLC using an HPX 87H column and ICS refractive index detector (RI 8120) with a mobile phase of 0.001 N H2 SO4 , flow rate of 0.8 ml/min, and column temperature of 25 ◦ C. The monosaccharide composition assays were performed in two independent experiments.
22 instrument (Germany) in the region of 4000–400 cm−1 at a resolution of 4 cm−1 and processed by the Bruker OPUS software. 2.5. Sequencing of the 16S rRNA gene of Lactobacillus sp. Ca6 strain The chromosomal DNA from the TN8 strain was extracted as previously described elsewhere [30]. The isolate was identified by sequencing the total sequence of the 16S rRNA gene amplified with primers S73 (50-AGAGTTTGAT CCTGGCTCAG-30) and S74 (50-AAGGAGGTGATCCAAGCC-30) [31]. The PCR products were purified using the Wizard SV Gel and PCR Clean-Up system (Promega). Sequencing was performed with an ABI 3100 Capillary DNA Sequencer (Applied Biosystems Inc., Foster City, CA, USA). 2.6. Acid and bile tolerance of Lactobacillus sp. Ca6 strain Acid tolerance was determined with MRS broth adjusted to pH 2–5 using hydrochloric acid. Samples of 100 ml were mixed with 10 ml of the pH-adjusted MRS broth, inoculated to a final Optical Density at 600 nm (OD600 nm) of 0.1, and incubated at 30 ◦ C. The OD600 nm values were recorded on an hourly basis for 8 h. Cultures grown on MRS broth at pH 6 served as controls. Bile tolerance was determined using MRS culture media with different concentrations (0.5, 1, 3 and 5%) of bovine bile (Sigma/Aldrich). Experiments were conducted without shaking, in 10 ml of MRS broth inoculated from 24 h-old cultures grown on the same medium, and diluted to a final OD600 nm of 0.1. The OD600 nm values were recorded on an hourly basis for 8 h. Cultures grown on MRS broth without bovine bile served as control. 2.7. Antibacterial activity of Lactobacillus Ca6 strain Antimicrobial activity was tested by the agar well-diffusion method. Ca6 strain culture filtrates were prepared by submitting supernatants from overnight centrifuged cultures to filtration, concentration with a rotavapor (10×) to a final volume of 1 ml, and filter sterilization (0.22 mm). The concentrates were assayed against Salmonella enterica ATCC 43972, Escherichia coli ATCC 8739, L. ivonavi BUG 496, and Staphylococcus aureus ATCC 6538 used as indicator bacteria. The inhibitory effects of the filtrates were assessed by monitoring the ability of the indicator bacteria to grow in the presence of the culture filtrates as previously described by Skytta and Mattila-Sandholm [32]. Three parallel wells were used for each culture filtrate and control sample test. 2.8. Susceptibility to antibiotics Antibiotic susceptibility assays were performed in triplicate and in accordance with method described by Charteris et al. [33]. Cells were grown in MRS broth at 30 ◦ C for 18 h to an OD600 nm of 0.8. MRS agar plates containing 25 ml of MRS agar were overlaid with 3 ml of soft agar (6%) containing 109 Colony Forming Unit per milliliter (CFU/ml). The plates were kept at room temperature for 1 h. Antibiotic discs (the API-ZYM system (BioMérieux, MontalieuVercieu, France)) were placed onto solid media and incubated under anaerobic conditions at 30 ◦ C for 24 h. Growth inhibition was read by measuring the diameter of the inhibition zones.
2.4. FT-IR spectroscopy 3. Results The major structural groups of the purified EPS were detected using Fourier-transformed infrared spectroscopy. The FTIR spectrum of EPS was obtained by the KBr method. The polysaccharide samples were pressed into KBr pellets at a sample-KBr ratio of 1:100. The FTIR spectra were recorded on a Bruker Vector
3.1. Screening of LAB producing EPS A total of 10 lactobacilli that were previously isolated from different intestinal segments of indigenous poultry in Tunisia by Ben
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Fig. 1. Evidence for the EPS of Ca6 strain on sucrose-supplemented MRS. Ca6 strain isolate grown on MRS agar plates containing 4% sucrose (w/v) (a) or 2% glucose (b) and at 30◦ C after 24–48 h of incubation.
Salah et al. [29] were screened for their abilities to produce EPS from sucrose as a substrate. Two isolates, namely Ca6 and TN9, were noted to exhibit stronger mucoïd phenotype forms (Fig. 1a) on MRS agar media containing 4% (w/v) sucrose as compared to those grown on MRS agar media containing glucose instead of sucrose (Fig. 1b). Both strains were selected for further optimization analyses. The results revealed that the highest EPS production yields were obtained with the Ca6 strain grown on MRS media supplemented with sucrose. Accordingly, the Ca6 strain was selected and maintained for further studies. The Ca6 strain generated an EPS production yield 2.4 g/l after 24 h of incubation at 30 ◦ C. The amount of EPS produced by Lactobacillus plantarum and Lactobacillus paraplantarum strains cultivated in MRS broth containing maltose ranged from 1.4 to 2.97 g/l [34]. Sample heat treatment is a crucial preliminary step in polysaccharide isolation processes seeking the complete recovery of EPS [35,36]. Badel et al. [37] previously reported that the specific carbon substrate differs from one species to another and that sucrose represents the best source for various lactobacilli. The results of the present study are in agreement with the ones previously reported by Naessens et al. [38]. In fact, the Oligo-and homopolysaccharides produced from sucrose by lactic acid bacteria (LAB) have received increasing attention mainly because of their promising potential for use as texturizing agents and prebiotics in several industrial applications [16,39].
3.3. FT-IR spectroscopy analysis Fourier transform-infrared spectroscopy has often been reported to offer a useful tool for monitoring the structural changes of biopolymers [40] The FTIR spectrum of the purified EPS presented in Fig. 4. The results revealed the presence of more complex peak patterns ranging from 3000 to 1200 cm−1 . The polysaccharides contained a significant number of hydroxyl groups, which exhibited an intense broad stretching peak at around 3307 cm−1 . The absorption in that region (Fig. 4) had the rounded trait typical of hydroxyl groups [41], suggesting that the substance was a polysaccharide. The FTIR spectra of the EPS revealed functional characteristic, including a broad-stretching hydroxyl group at 3307 cm−1 , two weak C H stretching peaks at 2966 and 2936 cm−1 corresponding to methyl and methylene groups. The strong absorption observed at 1652 cm−1 corresponded to the amide >C O stretch. Furthermore, the peak at 1404 cm−1 could be assigned to the >C O stretch of the COO− groups and the C O bond from COO− groups [9,42,43]. There was no peak around 1700–1775 cm−1 , suggesting that neither glucuronic acid nor diacyl ester was present in the EPS produced by Lactobacillus sp. Ca6 . The peak around 1652 cm−1 suggested the presence of the C O group [42], which was consistent with the results reported by Wang, et al. [5] A broad stretch of C O C, C O was observed at 1000–1200 cm−1 , thus suggesting the presence of carbohydrates [44]. The strongest absorption band at
3.2. Structure of EPS The results presented in Fig. 2 show the appearance of a one plug of monosaccharide having a retention factor of 0.28 (P1). The hydrolysis of the EPS produced by this strain led to the emergence of a plug having a retention factor of 0.62 corresponding to the same retention factor of the glucose (used as standard P4). The hydrolysis of the EPS was not total since part of the plug corresponding to the EPS persisted (RF = 0.28). The appearance of a plug corresponding to glucose indicated that the Ca6 EPS product was a homopolysaccharide consisting of a chain of glucose molecules. The monosaccharide composition of the EPS was investigated by HPLC analysis. The chromatograms showed a peak with a retention time of 7.5 min for non-hydrolyzed EPS (Fig. 3a), which was similar to the retention time of dextran used as standard (data not shown). The hydrolysis of the EPS produced by the Ca6 strain revealed the presence of a peak (0.76 min) whose retention time was similar to that of glucose (Fig. 3b), thus confirming that the EPS product by Lactobacillus strain Ca6 was a polymer of glucose.
Fig. 2. Monosaccharide composition of EPS produced by Lactobacillus sp. Ca6 in MRS supplemented sucrose (lane 1), non-hydrolyzed EPS (lane 2), hydrolyzed EPS Glucose (lane 3), fructose (lane 4), sucrose (lane 5) and xylose (lane 6) were used as standards.
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Fig. 3. Chromatograms of purified EPS. (a) Non hydrolyzed EPS; (b) hydrolyzed EPS.
1075 cm−1 was indicative that the substance was a polysaccharide [45] The FT-IR spectra of the polymer evidenced the presence of carboxyl groups, which can serve as binding sites for divalent cations [46] Moreover, the spectrum showed the presence of carboxyl and hydroxyl, the preferred groups for the flocculation process, a result that was similar to the findings previously reported for polyelectrolyte [47]. 3.4. Acidic and bile salt tolerance of the LAB isolates High acidity in the stomach and high bile concentrations in the proximal intestine of the host influence probiotic strain selection [48,49]. The pH in chicken GIT ranges from 2.5 to 4.7 [50], and ingestion can take 1–3 h, depending on the size of animal feed [29]. In this study, the Ca6 strain was tested for its ability to survive low
pH and high bile salt conditions. The Results indicated that Lactobacillus strain grew well under acid conditions at pH 4–6. Fig. 5 shows the OD600 nm values recorded during 8 h of Ca6 growth at pH 3. The Ca6 strain showed little or no decrease in OD600 nm for up to 5 h of growth at pH 4–6. It was also able to survive in acidic condition (pH 2 and 3) for 5 h, with OD values of 0.16, and 0.13, respectively. Previous reports have shown that only two of seven Lactobacillus acidophilus strains exhibited growth at pH 3 [51] and that only the L. acidophilus strain displayed high acid tolerance at pH 3 [52]. The results of the present study suggested that the Ca6 strain isolated from the gastro intestinal tract of poultry can be used as a probiotic additive. Bile tolerance is one of the properties required for Lactobacilli to survive in the small intestine, enabling them to play a role in the physiological function of this organ [53].
Fig. 4. Fourier-transformed infrared (FT-IR) spectrum of the exopolysaccharide produced by Lactobacillus sp. Ca6
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1,4 1,2 1
0% 0,50% 1% 3% 5%
1,2
0,8
1
0,6
OD (600 nm)
OD (600nm)
1,4
pH =6 pH =5 pH =4 pH =3 pH =2
0,4 0,2 0 0
1
2
3
4
5
6
7
8
9
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0,8 0,6 0,4
Times (h) Fig. 5. Time course of the cell growth of Lactobacillus sp. Ca6 isolated from gastrointestinal tract at various pH levels of 2–6. The experiment was performed in triplicate.
0,2 0
0
At 5% oxgall, the Ca6 strain showed a rapid decrease from 1.192 at 0% oxgall to 0.561 at 5% oxgall, with an OD value of 600 nm at 8 h (Fig. 6). A bile concentration of 3% is considered higher than the one normally observed in animal intestines. The results of this work, which are in agreement with those reported by Ramos et al. [54], suggest that the bacterial screening process involved in the search for novel probiotics should be performed at 5% bile concentration for at least 30 min. 3.5. Antibacterial activity The major properties of probiotic LAB lies in their inhibitory effect with regard to the growth of pathogenic bacteria [29]. Accordingly, several pathogenic bacteria were assayed as indicator bacteria, namely Gram negative bacteria, including E. coli ATCC 8739 and S. enterica ATCC 43972, and Gram positive bacteria,
1
2
3
4 5 Times(h)
6
7
8
9
Fig. 6. Time course of the cell growth of Lactobacillus sp. Ca6 at various oxgall levels of concentration (0, 0.5, 1, 3 and 5%). The experiment was performed in triplicate.
including Listeria ivanovii BUG 496, S. aureus ATCC 6538, Micrococcus luteus, and Enterobacter. Among the tested LAB strains, Lactobacillus Ca6 was noted to produce the largest inhibition zones, which ranged from 12 to 26 mm, against gram positive and gram negative pathogenic bacteria (Fig. 7). The antibacterial activity of LAB has often been attributed to the production of organic acids, with a consequent reduction in pH, or the production of hydrogen peroxide [29,55]. LAB could produce various compounds, such as organic acids, diacetyl, hydrogen peroxide, and bacteriocin or bactericidal proteins during lactic fermentations.
Fig. 7. inhibition zones against pathogenic bacteria A (E. coli DH5), B (Listeria ivanovii), C (Micrococcus luteus), D (Salmonella enterica), E (Staphylococcus aureus), and F (Enterobacter).
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Table 1 Antibiotic sensitivity of Lactobacillus sp. Ca6 . Antibiotics
Dose
Inhibition zones (mm)
Ampicillin Amoxillin Chloramphenicol Vancomycin Gentamycin
10 g 28 g 30 g 30 g 10 UI
23 25 25 10 20
3.6. Antibiotic susceptibilities A key requirement for probiotic strains is that they should not carry transmissible antibiotic resistance genes [29]. The antibiogram pattern of some antibiotics with regard to the Ca6 strain selected in the present work was investigated using various antibiotics (Table 1). The findings revealed that while the Ca6 strain was susceptible to -Lactam antibiotics (ampicillin), it showed resistance to antibiotics with gram-positive and other broad spectrum antibiotics (chloramphenicol), including vancomycin (Table 1). In fact, several LAB, including strains of Lactobacillus casei, Lactobacillus rhamnosus, L. plantarum, Pediococcus sp., and Leuconostoc sp., are resistant to vancomycin. This resistance is usually intrinsic, chromosomally encoded, and non transmissible [56]. Belkacem and Mebrouk [57] studied the antibiotic susceptibility of ten Lactobacillus stains isolated from broiler digestive tract and showed that most of the isolates exhibited high resistance to all the tested antibiotics. 3.7. Identification and phylogenetic analysis of Ca6 strain The total nucleotide sequence was determined from the whole 16S rRNA gene of strain Ca6 . The alignment of this sequence with previous 16S rRNA gene sequences available in the GeneBank database showed high similarity to Lactobacillus 16S rRNA reference genes. Based on the nucleotide sequence of the 16S rRNA gene of the Ca6 strain, the new isolate was identified as a Lactobacillus sp. 4. Conclusion The search for microbial exopolysaccharides has gained increasing attention in recent research. Lactobacillus sp. Ca6 presented in this study was noted to produce a homopolysaccharide, consisting of a polymer arrangement of glucose, which was confirmed by TLC and HPLC. The in vivo result showed that the Ca6 strain, namely Lactobacillus sp., was resistant to acidic (pH of 2) and bovine bile (5%) conditions, sensitive to several antibiotics, and able to exert antagonistic effects against enteric pathogenic bacteria. The probiotic and EPS production potential of this strain provide strong support for its promising candidacy for future industrial application, particularly in the dairy industry. Accordingly, further research, some of which is currently underway in our laboratories, is needed to investigate its applications in fermented dairy products. Acknowledgements The authors would like to express their sincere gratitude to Mr. Anouar Smaoui and Mrs. Hanen Ben Salem from the English Language Unit at the Faculty of Science of Sfax, Tunisia for their valuable proofreading and language polishing services. References [1] L.I. Shao, Z. Wub, H. Zhanga, W. Chena, L. Ai, B. Guoa, Carbohydr. Polym. 107 (2007) 51–56. [2] C. Arezoo, Nephrol. Dial. Transplant. 16 (2002) 17–20.
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Purification and characterization of a novel exopolysaccharides produced by Lactobacillus sp. Ca6 Imen Trabelsi, Sirine Ben Slima, Hela Chaabane, Ben Salah Riadh ∗ Laboratory of Microorganisms and Biomolecules (LMB), Centre of Biotechnology of Sfax, University of Sfax, Road of Sidi Mansour Km 6, P.O. Box 1177, Sfax 3018, Tunisia
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Article history: Received 1 December 2014 Received in revised form 16 December 2014 Accepted 19 December 2014 Available online 15 January 2015 Keywords: Exopolysaccharide (EPS) FTIR spectroscopy Probiotics
a b s t r a c t This study was undertaken to investigate the ability of ten lactic acid bacterial strains to produce exopolysaccharides (EPS) on MRS broth containing 4% sucrose. A maximum EPS production yield of 2.4 g/l was obtained by strain Lactobacillus sp. Ca6 . The results from thin layer chromatography (TLC) and high performance chromatography (HPLC) analyses showed that the EPS produced was a polymer of glucose. Further FTIR spectroscopic analysis revealed the presence of carboxyl, hydroxyl and amide groups corresponding to a typical EPS. In addition to EPS production, Lactobacillus sp. Ca6 displayed good probiotic properties (antimicrobial activities and sensitivity to several antibiotics) and resistance to acidic condition (pH 2) and 5% bile bovine. Overall, the findings indicate that this strain has a number of promising properties that make it a potential promising candidate for future application as a food additive. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The increased demand of natural polymers for various industrial applications in recent years has led to a renewed interest in the production of exopolysaccharides (EPS) from natural origins [1]. Several microorganisms, including lactic acid bacteria (LAB), fungi and plants, have an ability to synthesize extracellular polysaccharides as soluble or insoluble polymers [2–4]. Several EPS produced by natural microorganisms have been employed in the food, cosmetic, and pharmaceutical industries where they serve various functions, such as bioflocculants, bioabsorbents, heavy metal removal agents, and drug delivery agents [5]. Exopolysaccharides from microbial origins include dextrans, xanthan, gellan, pullulan, yeast glucans and bacterial alginates [4,6]. Various microbe-derived polysaccharides have been used as food additives, including xanthan from Xanthomonas campestris [7–9] and gellan from Pseudomonas elodea [10]. Several LABs have gained increasing attention in recent research particularly because of the textural properties they can impart to several biotechnological products and the “Qualified Presumption of Safety” (QPS) status they have acquired [11]. In fact, the implementation of functional cultures that “contribute to food safety and/or offer one or more organoleptic, technological and
∗ Corresponding author. Tel.: +216 74 87 04 51; fax: +216 74 87 04 51. E-mail addresses: riadh [email protected], [email protected] (B.S. Riadh). http://dx.doi.org/10.1016/j.ijbiomac.2014.12.045 0141-8130/© 2015 Elsevier B.V. All rights reserved.
nutritional, or health advantages” in the manufacture of fermented foods has gained growing attention in the last few decades [12,13]. In this context, several LABs, including Lactobacillus reuteri, have been reported for their ability to produce EPS for application as safe and healthy alternative bio-thickeners in various food products [14]. Based on their composition and biosynthesis mechanisms, EPS have been divided into two classes: heteropolysaccharides and homopolysaccharides. Heteropolysaccharides consist of multiple sugar types and are synthesized by the combined action of different types of glycosyltransferase enzymes [15]. Homopolysaccharides are generally synthesized from the sole sucrose substrate by the action of the sucrase enzyme [16]. Sucrase synthesizes polysaccharides consisting of either glucose (glucan) or fructose (fructan) residues [16]. In general, glucans and fructans can be used as viscosifying, stabilizing, emulsifying, sweetening, gelling, or waterbinding agents in the food, as well as in non-food, industry [17,18]. Sucrase genes of Leuconostoc, Streptococcus and L. reuteri strains, including L. reuteri ATCC 55730, have been characterized [19–23]. There is strong evidence in the literature that EPS have good emulsifying activity, a property that is highly desired for various food formulations [24,25]. EPS have also been shown to have important therapeutic activities [18,26,27]. The viscosity and biological activities of EPS depend on their primary structure, molecular mass, and sugar composition [28]. The amount of EPS production depends on the medium and cultural conditions used for microbial growth. The present study focused on
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the screening of ten lactic acid bacteria for their ability to produce EPS in sucrose-MRS broth. The EPS was separated, purified, and characterized by TLC, HPLC and FTIR-spectroscopy. The selected strain and its derivatives were evaluated in vitro as potential probiotics, including their resistance to acidic pH, bile bovine and antibiotics and their ability to inhibit pathogenic bacteria. 2. Materials and methods 2.1. Screening of produced EPS LABS from sucrose Ten LAB strains, which had previously been isolated in our laboratory from the gastrointestinal tract of indigenous poultry in Tunisia [29], were screened for their ability to produce EPS from sucrose. The samples were plated on De Man, Rogosa and Sharpe (MRS) agar media containing 4% (w/v) sucrose, 10 g/l tryptone, 10 g/l meat extract, 5 g/l yeast extract, 5 g/l sodium acetate, 2 g/l disodium phosphate, 2 g/l tri-ammonium citrate, 0.1 g/l MgSO4 , and 0.05 g/l MnSO4 (pH 6.5). After incubation for 24–48 at 30 ◦ C under anaerobic conditions, the plates were checked for the presence of colonies displaying a mucoïd aspect. 2.2. EPS extraction and purification The EPS was purified by a slightly modified method described by Garcîagaribay and Marshall [7]. In brief, strains Ca6 and TN9 were grown in 100 ml MRS-sucrose in an Erlenmeyer flask at 30 ◦ C for 48 h in anaerobic jars containing GasPak (Oxoid). The flasks were then taken out and heated at 100 ◦ C for 30 min to dissolve cell attached EPS and centrifuged at 12,000 rpm for 15 min. Crude EPS was precipitated by the addition of 3 volumes of chilled ethanol (95%) to the supernatant. After precipitation at 4 ◦ C (overnight) the sample was centrifuged at 12,000 rpm for 15 min, and the pellet was retained. The sample was redissolved in distilled water and lyophilized. The polymer dry mass of purified EPS was determined by measuring the mass of the precipitate. 2.3. Monosaccharide composition of EPS The purified EPS (2 mg) was hydrolyzed in 250 l of 2 M trifluoroacetic acid (TFA) at 100 ◦ C for 1 h. Excess TFA was removed by evaporation, and the hydrolysate was washed thoroughly with water and lyophilized. The lyophilized powder was dissolved in 100 l of water, and a 5-l aliquot was used for thin-layer chromatography (TLC). Migration was performed twice on a silica gel TLC plate (20 cm × 20 cm) using n-butanol:ethanol: water (2:1:1, v/v). Carbohydrates were visualized by heating the TLC plate after spraying with 5% (v/v) sulfuric acid in ethanol. Glucose, fructose, sucrose and xylose were used as standard monosaccharides. The supernatant was then used for the analysis of monosaccharides in the hydrolysate. A 20-l sample of the supernatant was added to 980 l of H2 SO4 and filtered through a 0.45 m pore size filter. Monosaccharide composition was analyzed by HPLC using an HPX 87H column and ICS refractive index detector (RI 8120) with a mobile phase of 0.001 N H2 SO4 , flow rate of 0.8 ml/min, and column temperature of 25 ◦ C. The monosaccharide composition assays were performed in two independent experiments.
22 instrument (Germany) in the region of 4000–400 cm−1 at a resolution of 4 cm−1 and processed by the Bruker OPUS software. 2.5. Sequencing of the 16S rRNA gene of Lactobacillus sp. Ca6 strain The chromosomal DNA from the TN8 strain was extracted as previously described elsewhere [30]. The isolate was identified by sequencing the total sequence of the 16S rRNA gene amplified with primers S73 (50-AGAGTTTGAT CCTGGCTCAG-30) and S74 (50-AAGGAGGTGATCCAAGCC-30) [31]. The PCR products were purified using the Wizard SV Gel and PCR Clean-Up system (Promega). Sequencing was performed with an ABI 3100 Capillary DNA Sequencer (Applied Biosystems Inc., Foster City, CA, USA). 2.6. Acid and bile tolerance of Lactobacillus sp. Ca6 strain Acid tolerance was determined with MRS broth adjusted to pH 2–5 using hydrochloric acid. Samples of 100 ml were mixed with 10 ml of the pH-adjusted MRS broth, inoculated to a final Optical Density at 600 nm (OD600 nm) of 0.1, and incubated at 30 ◦ C. The OD600 nm values were recorded on an hourly basis for 8 h. Cultures grown on MRS broth at pH 6 served as controls. Bile tolerance was determined using MRS culture media with different concentrations (0.5, 1, 3 and 5%) of bovine bile (Sigma/Aldrich). Experiments were conducted without shaking, in 10 ml of MRS broth inoculated from 24 h-old cultures grown on the same medium, and diluted to a final OD600 nm of 0.1. The OD600 nm values were recorded on an hourly basis for 8 h. Cultures grown on MRS broth without bovine bile served as control. 2.7. Antibacterial activity of Lactobacillus Ca6 strain Antimicrobial activity was tested by the agar well-diffusion method. Ca6 strain culture filtrates were prepared by submitting supernatants from overnight centrifuged cultures to filtration, concentration with a rotavapor (10×) to a final volume of 1 ml, and filter sterilization (0.22 mm). The concentrates were assayed against Salmonella enterica ATCC 43972, Escherichia coli ATCC 8739, L. ivonavi BUG 496, and Staphylococcus aureus ATCC 6538 used as indicator bacteria. The inhibitory effects of the filtrates were assessed by monitoring the ability of the indicator bacteria to grow in the presence of the culture filtrates as previously described by Skytta and Mattila-Sandholm [32]. Three parallel wells were used for each culture filtrate and control sample test. 2.8. Susceptibility to antibiotics Antibiotic susceptibility assays were performed in triplicate and in accordance with method described by Charteris et al. [33]. Cells were grown in MRS broth at 30 ◦ C for 18 h to an OD600 nm of 0.8. MRS agar plates containing 25 ml of MRS agar were overlaid with 3 ml of soft agar (6%) containing 109 Colony Forming Unit per milliliter (CFU/ml). The plates were kept at room temperature for 1 h. Antibiotic discs (the API-ZYM system (BioMérieux, MontalieuVercieu, France)) were placed onto solid media and incubated under anaerobic conditions at 30 ◦ C for 24 h. Growth inhibition was read by measuring the diameter of the inhibition zones.
2.4. FT-IR spectroscopy 3. Results The major structural groups of the purified EPS were detected using Fourier-transformed infrared spectroscopy. The FTIR spectrum of EPS was obtained by the KBr method. The polysaccharide samples were pressed into KBr pellets at a sample-KBr ratio of 1:100. The FTIR spectra were recorded on a Bruker Vector
3.1. Screening of LAB producing EPS A total of 10 lactobacilli that were previously isolated from different intestinal segments of indigenous poultry in Tunisia by Ben
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Fig. 1. Evidence for the EPS of Ca6 strain on sucrose-supplemented MRS. Ca6 strain isolate grown on MRS agar plates containing 4% sucrose (w/v) (a) or 2% glucose (b) and at 30◦ C after 24–48 h of incubation.
Salah et al. [29] were screened for their abilities to produce EPS from sucrose as a substrate. Two isolates, namely Ca6 and TN9, were noted to exhibit stronger mucoïd phenotype forms (Fig. 1a) on MRS agar media containing 4% (w/v) sucrose as compared to those grown on MRS agar media containing glucose instead of sucrose (Fig. 1b). Both strains were selected for further optimization analyses. The results revealed that the highest EPS production yields were obtained with the Ca6 strain grown on MRS media supplemented with sucrose. Accordingly, the Ca6 strain was selected and maintained for further studies. The Ca6 strain generated an EPS production yield 2.4 g/l after 24 h of incubation at 30 ◦ C. The amount of EPS produced by Lactobacillus plantarum and Lactobacillus paraplantarum strains cultivated in MRS broth containing maltose ranged from 1.4 to 2.97 g/l [34]. Sample heat treatment is a crucial preliminary step in polysaccharide isolation processes seeking the complete recovery of EPS [35,36]. Badel et al. [37] previously reported that the specific carbon substrate differs from one species to another and that sucrose represents the best source for various lactobacilli. The results of the present study are in agreement with the ones previously reported by Naessens et al. [38]. In fact, the Oligo-and homopolysaccharides produced from sucrose by lactic acid bacteria (LAB) have received increasing attention mainly because of their promising potential for use as texturizing agents and prebiotics in several industrial applications [16,39].
3.3. FT-IR spectroscopy analysis Fourier transform-infrared spectroscopy has often been reported to offer a useful tool for monitoring the structural changes of biopolymers [40] The FTIR spectrum of the purified EPS presented in Fig. 4. The results revealed the presence of more complex peak patterns ranging from 3000 to 1200 cm−1 . The polysaccharides contained a significant number of hydroxyl groups, which exhibited an intense broad stretching peak at around 3307 cm−1 . The absorption in that region (Fig. 4) had the rounded trait typical of hydroxyl groups [41], suggesting that the substance was a polysaccharide. The FTIR spectra of the EPS revealed functional characteristic, including a broad-stretching hydroxyl group at 3307 cm−1 , two weak C H stretching peaks at 2966 and 2936 cm−1 corresponding to methyl and methylene groups. The strong absorption observed at 1652 cm−1 corresponded to the amide >C O stretch. Furthermore, the peak at 1404 cm−1 could be assigned to the >C O stretch of the COO− groups and the C O bond from COO− groups [9,42,43]. There was no peak around 1700–1775 cm−1 , suggesting that neither glucuronic acid nor diacyl ester was present in the EPS produced by Lactobacillus sp. Ca6 . The peak around 1652 cm−1 suggested the presence of the C O group [42], which was consistent with the results reported by Wang, et al. [5] A broad stretch of C O C, C O was observed at 1000–1200 cm−1 , thus suggesting the presence of carbohydrates [44]. The strongest absorption band at
3.2. Structure of EPS The results presented in Fig. 2 show the appearance of a one plug of monosaccharide having a retention factor of 0.28 (P1). The hydrolysis of the EPS produced by this strain led to the emergence of a plug having a retention factor of 0.62 corresponding to the same retention factor of the glucose (used as standard P4). The hydrolysis of the EPS was not total since part of the plug corresponding to the EPS persisted (RF = 0.28). The appearance of a plug corresponding to glucose indicated that the Ca6 EPS product was a homopolysaccharide consisting of a chain of glucose molecules. The monosaccharide composition of the EPS was investigated by HPLC analysis. The chromatograms showed a peak with a retention time of 7.5 min for non-hydrolyzed EPS (Fig. 3a), which was similar to the retention time of dextran used as standard (data not shown). The hydrolysis of the EPS produced by the Ca6 strain revealed the presence of a peak (0.76 min) whose retention time was similar to that of glucose (Fig. 3b), thus confirming that the EPS product by Lactobacillus strain Ca6 was a polymer of glucose.
Fig. 2. Monosaccharide composition of EPS produced by Lactobacillus sp. Ca6 in MRS supplemented sucrose (lane 1), non-hydrolyzed EPS (lane 2), hydrolyzed EPS Glucose (lane 3), fructose (lane 4), sucrose (lane 5) and xylose (lane 6) were used as standards.
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Fig. 3. Chromatograms of purified EPS. (a) Non hydrolyzed EPS; (b) hydrolyzed EPS.
1075 cm−1 was indicative that the substance was a polysaccharide [45] The FT-IR spectra of the polymer evidenced the presence of carboxyl groups, which can serve as binding sites for divalent cations [46] Moreover, the spectrum showed the presence of carboxyl and hydroxyl, the preferred groups for the flocculation process, a result that was similar to the findings previously reported for polyelectrolyte [47]. 3.4. Acidic and bile salt tolerance of the LAB isolates High acidity in the stomach and high bile concentrations in the proximal intestine of the host influence probiotic strain selection [48,49]. The pH in chicken GIT ranges from 2.5 to 4.7 [50], and ingestion can take 1–3 h, depending on the size of animal feed [29]. In this study, the Ca6 strain was tested for its ability to survive low
pH and high bile salt conditions. The Results indicated that Lactobacillus strain grew well under acid conditions at pH 4–6. Fig. 5 shows the OD600 nm values recorded during 8 h of Ca6 growth at pH 3. The Ca6 strain showed little or no decrease in OD600 nm for up to 5 h of growth at pH 4–6. It was also able to survive in acidic condition (pH 2 and 3) for 5 h, with OD values of 0.16, and 0.13, respectively. Previous reports have shown that only two of seven Lactobacillus acidophilus strains exhibited growth at pH 3 [51] and that only the L. acidophilus strain displayed high acid tolerance at pH 3 [52]. The results of the present study suggested that the Ca6 strain isolated from the gastro intestinal tract of poultry can be used as a probiotic additive. Bile tolerance is one of the properties required for Lactobacilli to survive in the small intestine, enabling them to play a role in the physiological function of this organ [53].
Fig. 4. Fourier-transformed infrared (FT-IR) spectrum of the exopolysaccharide produced by Lactobacillus sp. Ca6
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1,4 1,2 1
0% 0,50% 1% 3% 5%
1,2
0,8
1
0,6
OD (600 nm)
OD (600nm)
1,4
pH =6 pH =5 pH =4 pH =3 pH =2
0,4 0,2 0 0
1
2
3
4
5
6
7
8
9
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0,8 0,6 0,4
Times (h) Fig. 5. Time course of the cell growth of Lactobacillus sp. Ca6 isolated from gastrointestinal tract at various pH levels of 2–6. The experiment was performed in triplicate.
0,2 0
0
At 5% oxgall, the Ca6 strain showed a rapid decrease from 1.192 at 0% oxgall to 0.561 at 5% oxgall, with an OD value of 600 nm at 8 h (Fig. 6). A bile concentration of 3% is considered higher than the one normally observed in animal intestines. The results of this work, which are in agreement with those reported by Ramos et al. [54], suggest that the bacterial screening process involved in the search for novel probiotics should be performed at 5% bile concentration for at least 30 min. 3.5. Antibacterial activity The major properties of probiotic LAB lies in their inhibitory effect with regard to the growth of pathogenic bacteria [29]. Accordingly, several pathogenic bacteria were assayed as indicator bacteria, namely Gram negative bacteria, including E. coli ATCC 8739 and S. enterica ATCC 43972, and Gram positive bacteria,
1
2
3
4 5 Times(h)
6
7
8
9
Fig. 6. Time course of the cell growth of Lactobacillus sp. Ca6 at various oxgall levels of concentration (0, 0.5, 1, 3 and 5%). The experiment was performed in triplicate.
including Listeria ivanovii BUG 496, S. aureus ATCC 6538, Micrococcus luteus, and Enterobacter. Among the tested LAB strains, Lactobacillus Ca6 was noted to produce the largest inhibition zones, which ranged from 12 to 26 mm, against gram positive and gram negative pathogenic bacteria (Fig. 7). The antibacterial activity of LAB has often been attributed to the production of organic acids, with a consequent reduction in pH, or the production of hydrogen peroxide [29,55]. LAB could produce various compounds, such as organic acids, diacetyl, hydrogen peroxide, and bacteriocin or bactericidal proteins during lactic fermentations.
Fig. 7. inhibition zones against pathogenic bacteria A (E. coli DH5), B (Listeria ivanovii), C (Micrococcus luteus), D (Salmonella enterica), E (Staphylococcus aureus), and F (Enterobacter).
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Table 1 Antibiotic sensitivity of Lactobacillus sp. Ca6 . Antibiotics
Dose
Inhibition zones (mm)
Ampicillin Amoxillin Chloramphenicol Vancomycin Gentamycin
10 g 28 g 30 g 30 g 10 UI
23 25 25 10 20
3.6. Antibiotic susceptibilities A key requirement for probiotic strains is that they should not carry transmissible antibiotic resistance genes [29]. The antibiogram pattern of some antibiotics with regard to the Ca6 strain selected in the present work was investigated using various antibiotics (Table 1). The findings revealed that while the Ca6 strain was susceptible to -Lactam antibiotics (ampicillin), it showed resistance to antibiotics with gram-positive and other broad spectrum antibiotics (chloramphenicol), including vancomycin (Table 1). In fact, several LAB, including strains of Lactobacillus casei, Lactobacillus rhamnosus, L. plantarum, Pediococcus sp., and Leuconostoc sp., are resistant to vancomycin. This resistance is usually intrinsic, chromosomally encoded, and non transmissible [56]. Belkacem and Mebrouk [57] studied the antibiotic susceptibility of ten Lactobacillus stains isolated from broiler digestive tract and showed that most of the isolates exhibited high resistance to all the tested antibiotics. 3.7. Identification and phylogenetic analysis of Ca6 strain The total nucleotide sequence was determined from the whole 16S rRNA gene of strain Ca6 . The alignment of this sequence with previous 16S rRNA gene sequences available in the GeneBank database showed high similarity to Lactobacillus 16S rRNA reference genes. Based on the nucleotide sequence of the 16S rRNA gene of the Ca6 strain, the new isolate was identified as a Lactobacillus sp. 4. Conclusion The search for microbial exopolysaccharides has gained increasing attention in recent research. Lactobacillus sp. Ca6 presented in this study was noted to produce a homopolysaccharide, consisting of a polymer arrangement of glucose, which was confirmed by TLC and HPLC. The in vivo result showed that the Ca6 strain, namely Lactobacillus sp., was resistant to acidic (pH of 2) and bovine bile (5%) conditions, sensitive to several antibiotics, and able to exert antagonistic effects against enteric pathogenic bacteria. The probiotic and EPS production potential of this strain provide strong support for its promising candidacy for future industrial application, particularly in the dairy industry. Accordingly, further research, some of which is currently underway in our laboratories, is needed to investigate its applications in fermented dairy products. Acknowledgements The authors would like to express their sincere gratitude to Mr. Anouar Smaoui and Mrs. Hanen Ben Salem from the English Language Unit at the Faculty of Science of Sfax, Tunisia for their valuable proofreading and language polishing services. References [1] L.I. Shao, Z. Wub, H. Zhanga, W. Chena, L. Ai, B. Guoa, Carbohydr. Polym. 107 (2007) 51–56. [2] C. Arezoo, Nephrol. Dial. Transplant. 16 (2002) 17–20.
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