Bangladesh J Pharmacol. 2016; 11: 573-576 VISUAL EXPERIMENT Extraction, isolation and purification of exopolysaccharide from lactic acid bacteria using ethanol precipitation method Vivek K. Bajpai1, Rajib Majumder1, Irfan A. Rather1 and Kangmin Kim2 1 Department of Applied Microbiology and Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk 712- 749, South Korea; 2 Division of Biotechnology, College of Environmental and Bioresource Sciences, Chonbuk National University, 79 Gobong-ro, Iksan-si-570-752, Joellabuk-do (Jeonbuk), South Korea. First two authors contributed equally Correspondence to Vivek K. Bajpai at vbiotech04@gmail.com and Kangmin Kim at activase@jbnu.ac.kr First Published: 2 June, 2016 DOI: 10.3329/bjp.v11i3.27170 ABSTRACT Lactic acid bacteria are classified ‘Generally Recognized As Safe’ (GRAS) with most effective potential to divert significant amount of fermentable sugars towards the biosynthesis of functional exopolysaccharide. Exopolysaccharides from lactic acid bacteria are receiving a renewed interest due to the claims of human health benefits, such as modulation of immune response system and more importantly in food and pharma industries as a texturizer, viscosifer, emulsifier and syneresis-lowering agent. Its purification methodology involves: a) Extraction of cell-free supernatant from lactic acid bacteria; b) Denature of protein using trichloroacetic acid; c) Ethanol precipitation; d) Dialysis; and e) Freeze drying. However, depending on nature of research, compounds can be further purified using scanning electron microscopy (SEM), infrared spectrum (IR); and nuclear magnetic resonance (NMR) spectral analyses. INTRODUCTION Synthesis of exopolysaccharides by lactic acid bacteria is well known phenomenon which exists as a cell- bound exopolysaccharides, adhering closely to the bacterial surface, and released exopolysaccharide that releases into the surrounding medium (Wang et al., 2011; Tallon et al., 2003). The exopolysaccharides are associated with microbial cells protection against the adverse environments including desiccation, toxic materials and osmotic stress (Wang et al., 2011). The exopolysaccharides are thought to play a significant role in the colonization of lactic acid bacteria to various ecosystems by facilitating the colonization of bacteria to intestinal mucosa, thus enhance the immunity of host (Gorska et al., 2010). Now-a-days the exopolysaccharides are used as bio-thickeners due to their stabilizing, emulsifying or gelling properties especially in the food industry (Wang et al., 2011). In addition, some of the exopolysaccharides produced by lactic acid bacteria may confer health benefits such as immunomodulatory, anti-tumor, anti-biofilm and antioxidant activities (Ramchandran and Shah, 2009; Gorska et al., 2010). Therefore, the objective of this study was to provide visual demonstration of step-by-step extraction, isolation and purification of functional exopolysaccharide from lactic acid bacteria. However, the production cost of these antimicrobials is expensive due to optimized media composition and laborious purification process in addition to low yield in food-grade medium. Therefore, cost-effective medium and purification processes are of great need. MATERIALS AND EQUIPMENTS 1. Bacteria growth media 2. Centrifuge tubes (depending on volume) 3. Trichloroacetic acid http://www.bioxbio.com/if/html/BANGL-J-PHARMACOL.html 4. Absolute ethanol 5. Dialysis membrane ((Mw cut-off 12,000–14,000 Da) and tubing closure 6. FalconTM 50 mL conical centrifuge tubes 7. Aluminum foil 8. Refrigerated centrifuge 9. Deionized water 10. Magnetic stirrer 11. Freeze dryer PREPARATION OF REAGENTS Bacteria growth media: Dissolve 56 g of MRS media (for lactic acid bacteria) in 1 liter of distilled water supplemented with 10 g glucose/liter, stirrer until it gets properly dissolved and autoclave it. Preparation of 80% stock of trichloroacetic acid: Dissolve 80 g trichloroacetic acid in 1 liter of sterile distilled water. Absolute ethanol: Purchased from Sigma (USA) VIDEO CLIPS Part 1: 7 min 42 sec Part 2: 3 min 36 sec METHOD Extraction, isolation and purification of exopolysaccharide 1. Lactic acid bacterium was cultured at 37ºC for 18~24 hours in MRS modified medium supplemented with 10% glucose. 2. After centrifugation (8,000 ×g for 20 min at 4ºC) of culture, the supernatant was collected and added with a final concentration of 14% trichloroacetic acid to denature the protein content. 3. The culture was further left for homogenization in a shaker (90 rpm) for 30-40 min followed by centrifugation at 8,000 ×g for 20 min at 4ºC. 4. The supernatant was then added to cold absolute ethanol (two-fold volume of supernatant) at 4ºC for 24 hours, followed by centrifugation at 8000 ×g at 4ºC for 20 min 5. These steps resulted in the isolation of crude precipitate. 6. Finally, the precipitate was dissolved in deionized water and dialyzed using Spectra/Por molecularporous tubular dialysis membrane for 24~48 hours. 7. The precipitate was then lyophilized in an IIShin freeze dryer (Korea). 8. The freeze-dried lyophilized powder of lactic acid bacterium was considered to be purified exopolysaccharides. 9. The purified exopolysaccharide was stored at -80ºC for further analysis. PROTOCOL 1. Grow the exopolysaccharide producing bacterium in 1 liter of MRS broth followed by incubation at 37 ºC for 18~24 hours. 2. Add 14% final volume concentration of trichloroacetic acid to denature the protein. 3. Incubate the culture broth at 37ºC for 30-40 min in a shaking incubator at 90 rpm. 4. Centrifuge the culture at 8,000 ×g for 20 min to pellet down the cells. 574 Bangladesh J Pharmacol 2016; 11: 573-576 https://www.youtube.com/v/PGDyHPEk5jY https://youtube.com/v/G8ZMZ4z1DMs 5. Collect the supernatant in a sterilize container. 6. Mix cold absolute ethanol in ratio of 1:2 (Sample supernatant: absolute ethanol). 7. Incubate the reaction mixture at 4ºC for 24~48 hours for precipitation. 8. Discard the supernatant and collect the precipitate (Repeat the process if any leftover). 9. Divide the precipitate into 50 mL falcon tubes equally depending on the volume 10. Centrifuge the precipitate at 8,000 ×g for 20 min to remove traces of media component. 11. Mix equal volume of deionized water to each tube containing the precipitate. 12. Pore the precipitate in dialysis membrane bags according to size and volume needed. 13. Perform dialysis of precipitate at 4ºC for 24~48 hours to remove traces of protein (change the distilled water of container 1-2 times during dialysis). 14. Collect the precipitate in a 50 mL falcon tube and keep the tube in a refrigerator at -80ºC for few hours. 15. Lyophilized the precipitate using a freeze drier which is a purified exopolysaccharide (Figure 1). 16. Depending on nature of work, further analysis such as SEM, IR or NMR analyses can be performed to identify specific exopolysaccharide. Figure 1: Flow chart of partial purification of functional exopolysaccharide DISCUSSION It is well known that Lactobacilli are useful microorganisms in dairy technology, along with documented history of use in foods. To date, a number of lactic acid bacteria strains have been screened for their ability to produce exopolysaccharides as well as their health beneficial effects. Moreover, production of exopolysaccharides is dependent on specific growth phase of bacteria and media components. Hence, favorable media composition especially carbon sources (glucose and/or fructose) and bacterial growth (early stationary phase) should be optimized in order to get better exopolysaccharides yield from lactic acid bacteria. Several concerns have been raised that during late stationary phase production of glycohydrolases may catalyze degradation of polysaccharides, resulting in decreased exopolysaccharides yield (Cerning et al., 1994). Degradation of exopolysaccharide production on prolonged incubation has been reported previously for other lactic acid bacteria strains (Degeest et al., 2001). Since regulation of the exopolysaccharides biosynthetic pathway in lactic acid bacteria is dependent on the carbohydrate/carbon sources added to the growth medium (Grobben et al., 1995), supplementation of these sources may result in the variations of exopolysaccharides recovery rate from different lactic acid bacteria strains. One of the Bangladesh J Pharmacol 2016; 11: 573-576 575 major drawbacks of exopolysaccharides purification is cost effectiveness and prolong-time consumption. However lactic acid bacteria producing exopolysaccharides have received considerable interest over the past couple of years because of their use in food and pharma industries as food preservatives as well as bio-thickener and viscosifer agents. REFERENCES Cerning J, Renard CMGC, Thibault JF, Bouillanne C, Landon M, Desmazeaud M, Topisirovic L. Carbon source requirements for exopolysaccharide production by Lactobacillus casei CG11 and partial structure analysis of the polymer. Appl Environ Microbiol. 1994; 60: 3914-19. Degeest B, Janssens B, De Vuyst L. Exopolysaccharide (EPS) biosynthesis by Lactobacillus sakei 0-1: Production kinetics, enzyme activities and EPS yields. J Appl Microbiol. 2001; 91: 470-47. Grobben GJ, Sikkema J, Smith MR, De Bont JAM. Production of extracellular polysaccharides by Lactobacillus delbrueckii ssp. bulgaricus NCFB 2772 grown in a chemically defined medium. J Appl Bacteriol. 1995; 79: 103-07. Gorska S, Jachymek W, Rybka J, Strus M, Heczko PB, Gamian A. Structural and immunochemical studies of neutral exopolysaccharide produced by Lactobacillus johnsonii 142. Carbohydr Res. 2010; 345: 108-14. Ramchandran L, Shah NP. Effect of exopolysaccharides and inulin on the proteolytic, angiotensin-I- converting enzyme and α-glucosidase-inhibitory activities as well as on textural and rheological properties of low-fat yogurt during refrigerated storage. Dairy Sci Technol. 2009; 89: 583-600. Tallon R, Bressollier P, Urdaci MC. Isolation and characterization of two exopolysaccharides produced by Lactobacillus plantarum EP56. Res Microbiol 2003; 154: 705-12. Wang CL, Huang TH, Liang TW, Fang CY, Wang SL. Production and characterization of exopoly- saccharides and antioxidant from Paenibacillus sp. TKU023. N Biotechnol. 2011; 28: 559-65. PRECAUTION Before adding sample to dialysis membrane, it is highly recommended to wash the membrane with ultrapure water or buffer for at least 30 min to remove contaminants such as sulfur, heavy metals and glycerol to avoid any possible effects of these components on sample. In addition, the rate of dialysis also depends on surface area of the membrane as well as the concentration of molecule. Therefore, it is recommended using bigger flasks to carry out dialysis on magnetic stir with regular distilled water and/or suitable buffer exchange. It is important to repeat sample dialysis process 2-3 times in order to get rid of undesirable protein from exopolysaccharides sample. More removal of protein content from sample during dialysis will provide more purified exopolysaccharides with diverse functional ability. Also it is advisable to repeat ethanol precipitation process 2-3 times to get release of maximum amount of exopolysaccharides from cell-free bacterial supernatant. Lyophilisation of exopolysaccharides is crucial stage, hence, precautions are needed while drying it completely so a further desired analysis could be performed in an easy way. 576 Bangladesh J Pharmacol 2016; 11: 573-576 Your feedback about this paper 1. Number of times you have read this paper 2. Number of times you have seen the video clip 3. Quality of paper 4. Your comments Text1: Dropdown2: [Click] Dropdown3: [0] Dropdown4: [0] DatePrinted: This article was downloaded by you on: Jun 02, 2016