All of the chemicals used were of analytical reagent grade. Sephadex G75 was obtained from Bangalore Genie Private Ltd (Bangalore, India). The remaining chemicals were procured from Merck Specialities Private Ltd (Mumbai, India).
Source of culture
KM1 bacterial isolate showing high EPS content was isolated from pooled human milk and was maintained on MRS agar and used throughout the study (Fig. 2a). The culture was maintained by biweekly transfers into sterile litmus at 1% level by inoculated at 37 °C for 24 h and held at − 4 °C between transfers. The sequence analysis of 16S ribosomal RNA gene technique (16S rRNA) was employed for identification of isolate KM1. Then the sequence homologies were analysed by comparative studies using “The National Centre for Biotechnology Information (NCBI)” using web link (http://www.ncbi.nlm.nih.gov/) and Basic Alignment Search Tool (BLAST). The registered accession number of Lactobacillus paraplantarum KM1 is KX671558.
Culture conditions for EPS production
Eighteen-hour-old inoculum (108 CFU/ml) was obtained in MRS medium after inoculating under static condition at 37 °C. EPS production (Fig. 2a) was achieved in optimized MRS medium (pH 6.5) with addition of lactose (1.5%) and ammonium sulphate (2%) at 35 °C for 32 h [1, 11].
Purification
Centrifugation at 12,000g for 20 min at 4 °C was done for 32-h culture, and 0.22-μm membrane filter (Millipore Corp., Cork, Ireland) was used to filter supernatant. Two volumes of 10% (w/v) trichloroacetic acid were added to the filtrate to obtain protein precipitates, and after keeping it overnight at 4 °C under static conditions, it was recentrifuged at 25,000g for 20 min. Four volumes of prechilled 95% (v/v) ethanol were added to the supernatant, further keeping it for 24 h at 4 °C and centrifuging at 25,000g for 20 min at 4 °C to obtain polysaccharide precipitates. The pellet was further lyophilized to make the EPS dry powder. The EPS powder was dissolved in 5-ml distilled water and dialysed using a 14-kDa cut-off dialysis membrane (Himedia, Mumbai, India). The EPS which was dialysed was relyophilized and assessed for carbohydrate content. Carbohydrate content was spectrophotometrically analysed at 490 nm (750 Lambda Double Beam UV–Vis Spectrophotometer; Perkin Elmer, Shelton, CT, USA) following the phenol–sulphuric acid method [16]. Subsequentially, deproteinized EPS was purified by Sephadex G75 chromatography. Sephadex G75 column (2.5 × 100 cm) was used to mount deproteinized EPS solution (10 mg/ml, 3 ml) as per the reported method [17]. Distilled water elute (3 ml/2 min) was collected automatically, and the carbohydrate content was determined using the phenol-sulphuric acid method with glucose as a standard [16]. The polysaccharide fractions obtained were then pooled, concentrated and lyophilized (Fig. 2b) for further study.
Characterization
Effect of temperature
The effect of temperature change on purified EPS from − 20 to 100 °C was studied after 1 h of incubation, and the carbohydrate content was determined using the phenol-sulphuric acid method with glucose as a standard.
Effect of pH
The purified EPS with pH varying from 5.5 to 8 was studied for 1 h of incubation period, and the carbohydrate content was determined using the phenol-sulphuric acid method with glucose as a standard.
Stability
The stability study of purified EPS from L. paraplantarum KM1 was done at room temperature for time interval of 0, 1, 2, 3, 5, 7, 15 and 30 days, and the carbohydrate content was determined using the phenol-sulphuric acid method with glucose as a standard.
Antimicrobial test
To study antagonistic potential, food borne/spoilage causing bacteria viz., Staphylococcus aureus IGMC, Listeria monocytogens MTCC 839, Leucononstoc mesenteroids MTCC 107 Enterococcus faecalis MTCC 2729, Clostridium perfringens MTCC 1739 and Bacillus cereus CRI, were used. The test strains were arranged from the Institute of Microbial Technology (IMTECH, Chandigarh, India), Indira Gandhi Medical College (IGMC, Shimla, H.P., India) and Central Research Institute (CRI, Kasauli, H.P., India). All these test strains were revived in nutrient agar medium twice for 24 h at 37 °C before performing experiments, as all these indicators were preserved in 40% glycerol at – 20 °C. Purified EPS from L. paraplantarum KM1 showed antimicrobial activity against various test organisms @ 1 O.D culture using agar well diffusion assay. The diameter of zone of inhibition extending laterally around the well was measured, and a clear zone of 1 mm or more was considered positive inhibition.
Solubility
Solubility of L. paraplantarum KM1 EPS in water was determined by following the procedure [18]. A suspension was made by dissolving EPS at rate 50 mg/ml in water with continuous agitation at 35 °C for 24 h. This was followed by centrifugation at 5000×g for 15 min, and the collected supernatant (0.2 ml) was precipitated with 3 volumes of ethanol. Again, EPS in the form of precipitate was recovered by centrifugation at 10,000×g for 5 min. The resultant material was vacuum dried at 50 °C, and the difference in weight was recorded. The solubility was calculated using the given formula:
$$ \mathrm{Solubility}\ \left(\%\right)=\frac{\mathrm{Total}\ \mathrm{carbohydrate}\ \mathrm{concentration}\ \mathrm{in}\ \mathrm{supernatant}\ }{\mathrm{weight}\ \mathrm{of}\ \mathrm{sample}\ \left(\mathrm{dry}\ \mathrm{weight}\ \mathrm{basis}\right)}\times 100 $$
Water-holding capacity
Purified EPS was characterized for water-holding capacity (WHC) by suspending 0.2-g sample in 10 ml of deionized water on a vortex mixer [18]. Dispersed material was centrifuged at 10,000 rpm for 25 min. Unbound water that was not held by EPS material was discarded. The entire EPS material was dropped on the pre weight filter paper for complete drainage of water. The weight of EPS thus precipitated was recorded. The percentage of WHC was calculated using the following expression:
$$ \mathrm{WHC}\ \left(\%\right)=\frac{\mathrm{Total}\ \mathrm{sample}\ \mathrm{weight}\ \mathrm{after}\ \mathrm{water}\ \mathrm{absorption}}{\mathrm{weight}\ \mathrm{of}\ \mathrm{sample}\ \left(\mathrm{dry}\ \mathrm{weight}\ \mathrm{basis}\right)}\times 100 $$
Analysis of EPS hydrolysates
HPLC separation of monosaccharide anthranilic acid derivatives was done to identify their composition as described by Anumula [19] using the services provided by NIPER, Mohali, Punjab, India. Analysis of monosaccharide derivatives was aided by reversed-phase C18 column (ZORBAX 300 SB-C18, 5 ml, 4.6 × 250 mm, Agilent Technologies Pvt. Ltd, Alexandra Point, Singapore) of Agilent 1000 series HPLC (Model no. 1100; Agilent Technologies) equipped with a UV detector. Mobile phase system consisted of a gradient programme of solvent A and solvent B combination following the method described by Anumula [19]. Solvent A contained (v/v) 0.5% phosphoric acid, 0.2% 1-butylamine and 1% tetrahydrofuran mixed in water, while solvent B comprised in (v/v) acetonitrile (50%) and solvent A (50%). The separations were realized using a flow rate of 1 ml/min at 24 °C and 20 μl of each sample was injected at a time.
$$ \mathrm{Concentration}\ \mathrm{of}\ \mathrm{Sugars}\ \left(\frac{\mathrm{mg}}{\mathrm{ml}}\right)=\frac{\mathrm{Area}\ \mathrm{of}\ \mathrm{sample}}{\mathrm{Area}\ \mathrm{of}\ \mathrm{standard}}\times \mathrm{Standard}\ \mathrm{dilution} $$
MALDI-ToF mass spectrometry
TFA (100 mmol/ml) was used to partially degrade EPS, and aqueous ethanol [80% (v/v)] was helpful in removing acid resistant material from the digest by diluting it. The ethanol soluble oligosaccharides were then lyophilized. The 1-ml deionized water was used to dissolve 1 mg of oligosaccharide. The sample was mixed with equal volume of 2,5-dihydroxybenzoic acid (10 mg/ml) as matrix, prior to MALDI analysis. MALDI analysis was done on an Applied Biosystem Voyager-DE PRO MALDI ToF mass spectrometer with a nitrogen laser (337 nm) operated in an accelerating voltage (20 kV). Every spectrum was obtained in the positive ion reflector mode as an average of 100 laser shots. External calibration of the data was done using angiotensin and ACTH (Applied Biosystem, Carlsbad, CA, USA). The reproducibility of each spectrum was checked five times by comparing it with the prepared sample.
Surface analysis of purified L. paraplantarum KM1 EPS
Surface analysis of purified EPS was studied by scanning electron microscope (SEM) at different magnifications. EPS was fixed on aluminium stub and gold sputtered. The sample was examined under VEGA TESCAN SEM (Brno, Czech Republic) using services provided NIPER, Mohali, Punjab, India.
Cytotoxicity evaluation
The extracted purified EPS was evaluated for cytotoxicity using mammalian epithelial cell line (HEp-2C) by MTT assay for their safety usage pattern. HEp-2C cells were maintained in Earle’s Minimum Essential Medium (Sigma, USA), supplemented with 10% foetal calf serum (Biosera, UK), 1% non-essential amino acids (Sigma, USA), 2 M l-glutamine (Sigma, USA), 100 μg/ml streptomycin (Sigma, USA) and 100 IU/ml penicillin. By seeding cells in 96-well culture plates (Polylabo, France), MTT assay [20] was repeated three times for each EPS concentration, at the rate of 2.104 cells/well, and treated with increasing concentrations of EPS (0, 100, 200, 300 and 400 μg/ml) for 24 h. The MTT solution (150 μl, 5 mg/ml) was mixed in the culture medium, poured into culture wells, and then, plates were incubated for 3 h at 37 °C. Supernatant was discarded, and the absorbance was measured using an ELISA reader at 540 nm with 690 nm reference. Dose-response curves were computer plotted after converting the mean data values to percentages of the control response. Cultures were then incubated with EPS for 24 h, and the dose of EPS which gave 50% inhibition of live cells (IC50) was derived from the plotted data by linear extrapolation. The data were expressed as mean ± standard deviation (SD) from at least three independent determinations (triplicates) for each experimental point.
Statistical analysis
Statistical tool applied for the analysis of data was central composite design (CRD).
