A commonly applied chemical-purification method is applied [21 (link)]. This chemical extraction method does not influence the alginate gelling condition [22 (link)]. The purification procedure was categorized into six distinct steps. After each step a sample was taken to assess the immunostimulatory capacity in order to gain insight in the efficacy of removing immunostimulatory contaminants in the alginate.
Step 0: a sample from crude non-purified alginate was taken.
Step 1: crude sodium alginate was dissolved at 4 °C in a 1 mM sodium ethylene glycol tetraacetic acid (EGTA) solution to a 1% solution under constant stirring. Subsequently, the solutions were filtered over successively 5.0, 1.2, 0.8, and 0.45 μm filters (Whatman®, Dassel, Germany). During this filtration step, all visible aggregates were removed.
Step 2: the pH of the solution was lowered to 3.5 by addition of 2 N HCl + 20 mM NaCl. The solution was kept on ice to prevent hydrolysis of alginate. The next step was to slowly lower the pH from 3.5 to 2.0. This is associated with gradual precipitation of alginate as alginic acid [43 ]. Routinely, the solutions were brought at a pH of 2.0 and subsequently filtered over a Buchner funnel (pore size 1.5 mm) to wash out non-precipitated contaminants. To extend the washout of non-precipitated contaminants, the precipitate was brought in 0.01 N HCl + 20 mM NaCl, vigorously shaken, and filtered again over the Buchner funnel. This washing procedure was performed three times and another sample was taken.
Step 3: proteins were removed by extraction with chloroform/butanol [44 ]. The alginic acid was suspended in 100 mL of 0.01 N HCl + 20 mM NaCl and supplemented with chloroform (20 mL at each 100 mL alginate solution) and 1-butanol (5 mL at each 100 mL alginate solution). The mixture was vigorously shaken for 30 min and filtered over the Buchner funnel. This chloroform/butanol extraction was performed three times and a third sample was obtained after the last extraction.
Step 4: the alginic acid was brought in water and slowly dissolved by gradually raising the pH to 7.0 by slow addition of 0.5 N NaOH + 20 mM NaCl over a period of at least one hour. The alginate solution obtained was subjected to a chloroform/butanol extraction to remove those proteins which can only be dissolved in chloroform/butanol at neutral pH [44 ]. The solution was vigorously shaken in a mixture of chloroform (20 mL at each 100 mL alginate solution) and 1-butanol (5 mL at each 100 mL alginate solution) for 30 min. The mixture was centrifuged for 5 min at 1800 rpm, which induced the formation of a separate chloroform/butanol phase, which was removed by aspiration. The extraction was repeated once and then a sample was taken.
Step 5: the last step is precipitation of the alginate with ethanol [43 ]. To each 100 mL of alginate solution we added 200 mL of absolute ethanol. After an incubation period of 10 min, all alginate had precipitated. The alginate was filtered over the Buchner funnel and washed two times with absolute ethanol and a sample was obtained.
Step 6: subsequently, the alginate was washed three times with ethylether and the last sample of purified alginate was taken (Figure 6 ).
All the samples of alginate were freeze-dried (Freezone 2.5 Plus, Labconco, Kansas, MO, USA) overnight for immunostimulation and enzyme-linked immunosorbent assays (ELISAs).
Step 0: a sample from crude non-purified alginate was taken.
Step 1: crude sodium alginate was dissolved at 4 °C in a 1 mM sodium ethylene glycol tetraacetic acid (EGTA) solution to a 1% solution under constant stirring. Subsequently, the solutions were filtered over successively 5.0, 1.2, 0.8, and 0.45 μm filters (Whatman®, Dassel, Germany). During this filtration step, all visible aggregates were removed.
Step 2: the pH of the solution was lowered to 3.5 by addition of 2 N HCl + 20 mM NaCl. The solution was kept on ice to prevent hydrolysis of alginate. The next step was to slowly lower the pH from 3.5 to 2.0. This is associated with gradual precipitation of alginate as alginic acid [43 ]. Routinely, the solutions were brought at a pH of 2.0 and subsequently filtered over a Buchner funnel (pore size 1.5 mm) to wash out non-precipitated contaminants. To extend the washout of non-precipitated contaminants, the precipitate was brought in 0.01 N HCl + 20 mM NaCl, vigorously shaken, and filtered again over the Buchner funnel. This washing procedure was performed three times and another sample was taken.
Step 3: proteins were removed by extraction with chloroform/butanol [44 ]. The alginic acid was suspended in 100 mL of 0.01 N HCl + 20 mM NaCl and supplemented with chloroform (20 mL at each 100 mL alginate solution) and 1-butanol (5 mL at each 100 mL alginate solution). The mixture was vigorously shaken for 30 min and filtered over the Buchner funnel. This chloroform/butanol extraction was performed three times and a third sample was obtained after the last extraction.
Step 4: the alginic acid was brought in water and slowly dissolved by gradually raising the pH to 7.0 by slow addition of 0.5 N NaOH + 20 mM NaCl over a period of at least one hour. The alginate solution obtained was subjected to a chloroform/butanol extraction to remove those proteins which can only be dissolved in chloroform/butanol at neutral pH [44 ]. The solution was vigorously shaken in a mixture of chloroform (20 mL at each 100 mL alginate solution) and 1-butanol (5 mL at each 100 mL alginate solution) for 30 min. The mixture was centrifuged for 5 min at 1800 rpm, which induced the formation of a separate chloroform/butanol phase, which was removed by aspiration. The extraction was repeated once and then a sample was taken.
Step 5: the last step is precipitation of the alginate with ethanol [43 ]. To each 100 mL of alginate solution we added 200 mL of absolute ethanol. After an incubation period of 10 min, all alginate had precipitated. The alginate was filtered over the Buchner funnel and washed two times with absolute ethanol and a sample was obtained.
Step 6: subsequently, the alginate was washed three times with ethylether and the last sample of purified alginate was taken (
All the samples of alginate were freeze-dried (Freezone 2.5 Plus, Labconco, Kansas, MO, USA) overnight for immunostimulation and enzyme-linked immunosorbent assays (ELISAs).
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