HOPE Lab Internal Training Protocol
This protocol details the extraction and purification of exopolysaccharides (EPS) from Lactobacillus strains grown in MRS broth. The method combines ethanol precipitation with trichloroacetic acid (TCA) deproteinization to yield high‑purity EPS suitable for biochemical and functional characterization.
1.1 Modified MRS Broth (per 1.0 Liter)
Prepare the medium using ultrapure water (18.2 MΩ·cm). Weigh and dissolve the following components completely:
Proteose peptone No. 3: 10.0 g
Beef extract: 10.0 g
Yeast extract: 5.0 g
D‑(+)‑Glucose: 20.0 g
Tween 80: 1.0 mL
Ammonium citrate: 2.0 g
Sodium acetate trihydrate: 5.0 g
Dipotassium hydrogen phosphate (K₂HPO₄): 2.0 g
Magnesium sulfate heptahydrate (MgSO₄·7H₂O): 0.20 g
Manganese sulfate monohydrate (MnSO₄·H₂O): 0.05 g
Adjust the pH to 6.5 using 1.0 M HCl or 1.0 M NaOH. Sterilize by autoclaving at 121 °C for 15.0 minutes. For solid agar media, add 15.0 g of bacteriological agar per liter before autoclaving.
1.2 Reagents for Extraction
Trichloroacetic acid (TCA) stock solution (80.0% w/v): Dissolve 80.0 g of TCA in ultrapure water and bring the final volume to 100.0 mL. Store at 4.0 °C in an amber bottle.
Absolute ethanol (≥99.5% purity): Pre‑chill a sufficient volume (at least 3.0 L per 1.0 L of culture) to −20.0 °C for a minimum of 12.0 hours before use.
Ultrapure water (18.2 MΩ·cm), pre‑chilled to 4.0 °C.
Dialysis tubing (flat‑width MWCO 12–14 kDa): Prepare by boiling in 2.0% (w/v) sodium bicarbonate and 1.0 mM EDTA for 10.0 minutes, rinsing thoroughly with ultrapure water, and storing in 20.0% (v/v) ethanol at 4.0 °C until use.
Step 1: Inoculum Preparation (Seed Culture)
Revive the Lactobacillus strain from a −80.0 °C frozen stock by streaking onto MRS agar plates.
Incubate the plates at 37.0 °C for 24.0 hours under static, microaerophilic conditions.
Pick a single, well‑isolated colony and aseptically transfer it into 10.0 mL of sterile MRS broth.
Incubate at 37.0 °C for 18.0 hours under static conditions.
Transfer 2.0 mL of this 18‑hour culture into 100.0 mL of fresh, sterile MRS broth.
Incubate at 37.0 °C for 18.0 hours under static conditions.
Measure the optical density at 600 nm (OD₆₀₀). Use the culture only when the OD₆₀₀ reaches exactly 0.800 (measured against a sterile MRS blank).
Step 2: EPS Production Fermentation
Inoculate 1.0 L of sterile MRS broth (pre‑equilibrated to 37.0 °C) with 20.0 mL (2.0% v/v) of the seed culture (OD₆₀₀ = 0.800).
Incubate the culture under static, microaerophilic conditions at 37.0 °C for exactly 48.0 hours.
Critical: Do not shake or agitate the culture during fermentation to prevent shear degradation of the EPS matrix.
Step 3: Heat Inactivation of Enzymes
Immediately upon completion of the 48.0‑hour fermentation, place the entire culture flask into a pre‑heated water bath set at 100.0 °C.
Heat for exactly 15.0 minutes to inactivate endogenous glycosidases and EPS‑degrading enzymes.
Remove the flask and allow the culture to cool passively to room temperature (25.0 °C).
Step 4: Removal of Bacterial Cells
Dispense the cooled culture into centrifuge bottles (balanced).
Centrifuge at 10,000 × g for 20.0 minutes at 4.0 °C.
Carefully decant the clear supernatant into a fresh sterile glass beaker. Discard the cellular pellet.
Step 5: Protein Removal via TCA Precipitation
Measure the exact volume of the recovered cell‑free supernatant (e.g., 950 mL).
Add the 80.0% (w/v) TCA stock solution dropwise while stirring gently with a magnetic stir bar to achieve a final concentration of 4.0% (w/v). Calculation example: For 950 mL of supernatant, add 50.0 mL of 80% TCA to reach 4.0% final.
Continue stirring gently at room temperature for exactly 30.0 minutes.
Transfer the mixture to an ice‑water bath and incubate for exactly 30.0 minutes to complete protein precipitation.
Centrifuge the mixture at 10,000 × g for 20.0 minutes at 4.0 °C.
Carefully collect the clear supernatant, ensuring absolutely no disturbance to the precipitated protein pellet. Transfer the supernatant to a new vessel.
Step 6: Primary EPS Precipitation
Measure the exact volume of the protein‑free supernatant.
Slowly add 3.0 volumes of pre‑chilled (−20.0 °C) absolute ethanol along the inner wall of the vessel while swirling the liquid gently to ensure immediate and thorough mixing. Do not vortex.
Seal the vessel and incubate at −20.0 °C for 16.0 hours (overnight) to precipitate the crude EPS.
Step 7: Recovery of Crude EPS Pellet
Centrifuge the precipitated mixture at 10,000 × g for 20.0 minutes at 4.0 °C.
Carefully pour off and discard the ethanolic supernatant.
Invert the centrifuge tube on a sterile absorbent pad for 2.0 minutes to drain residual liquid. Retain the white/gelatinous crude EPS pellet.
Step 8: Washing (Secondary Precipitation)
Resuspend the crude EPS pellet completely in 10.0 mL of ultrapure water by pipetting gently.
Add 30.0 mL of pre‑chilled (−20.0 °C) absolute ethanol (3.0 volumes) slowly with gentle swirling.
Incubate at −20.0 °C for exactly 2.0 hours.
Centrifuge at 10,000 × g for 20.0 minutes at 4.0 °C.
Discard the supernatant completely. This washing step effectively removes co‑precipitated salts and low‑molecular‑weight metabolites.
Step 9: Dissolution
Dissolve the washed EPS pellet in 25.0 mL of ultrapure water.
Stir the solution gently using a magnetic stirrer at 4.0 °C for 12.0 hours at 100 rpm to ensure complete dissolution of the polysaccharide.
Step 10: Dialysis
Transfer the dissolved EPS solution into the prepared dialysis tubing (MWCO 12–14 kDa), leaving sufficient headspace for buffer exchange.
Dialyze against 3.0 L of ultrapure water at 4.0 °C with continuous stirring at 80 rpm.
Replace the external dialysis water with fresh 3.0 L of ultrapure water at exactly 12.0 hours, 24.0 hours, and 36.0 hours.
Terminate the dialysis process at exactly 48.0 hours from the start.
Step 11: Lyophilization (Freeze‑Drying)
Transfer the dialyzed retentate from the tubing into a pre‑weighed, sterile lyophilization flask. Record the volume (typically ~25–30 mL).
Snap‑freeze the sample at −80.0 °C for exactly 6.0 hours until completely solid.
Connect the flask to the freeze‑dryer. Set the shelf temperature to −50.0 °C and maintain the chamber pressure at <0.10 mbar.
Lyophilize for exactly 48.0 hours.
Step 12: Storage and Yield Determination
Release the vacuum and immediately seal the flask.
Weigh the flask to determine the dry weight of the purified EPS.
Transfer the white, fluffy, fibrous powder into sterile, airtight glass vials.
Store the vials at −20.0 °C under desiccation (with silica gel) until further characterization.
MRS Glucose Concentration: 20.0 g/L
Initial MRS pH: 6.5
Inoculum Size: 2.0% (v/v)
Seed Culture OD₆₀₀: 0.800
Fermentation Temperature: 37.0 °C
Fermentation Time: 48.0 hours
Heat Inactivation: 100.0 °C / 15.0 min
Cell Removal Centrifugation: 10,000 × g / 20.0 min / 4.0 °C
Final TCA Concentration: 4.0% (w/v)
TCA Mixing Time (Room Temp): 30.0 min
TCA Ice Incubation Time: 30.0 min
Protein Removal Centrifugation: 10,000 × g / 20.0 min / 4.0 °C
Ethanol Precipitation Volumes: 3.0 volumes
Ethanol Temperature: −20.0 °C
Primary Precipitation Duration: 16.0 hours
Secondary Wash Precipitation Duration: 2.0 hours
Dissolution Water Volume: 25.0 mL
Dissolution Time/Temperature: 12.0 hours / 4.0 °C
Dialysis MWCO: 12–14 kDa
Dialysis Buffer Volume (per exchange): 3.0 L
Dialysis Exchange Intervals: 12.0, 24.0, 36.0 hours
Total Dialysis Duration: 48.0 hours
Freezing Temperature / Duration: −80.0 °C / 6.0 hours
Lyophilization Shelf Temperature: −50.0 °C
Lyophilization Chamber Pressure: <0.10 mbar
Lyophilization Duration: 48.0 hours
Final Storage Temperature: −20.0 °C
HOPE Lab Internal Training Notice
This protocol is provided for internal lab training and standardization. For publication‑quality EPS characterization, additional purification steps (e.g., ion‑exchange chromatography) and compositional analyses may be required. Always consult the latest literature or manufacturer guidelines when adapting this protocol to new strains or downstream applications.
HOPE Lab Internal Training Document
Laboratory for Health, Omics and Pathway Exploration (HOPE Lab)
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