Alginic Acid

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).

LMW fucoidan extracted from Laminaria japonica were collaboratively manufactured by Hi-Q Marine Biotech International Ltd. (New Taipei City, Taiwan). Seaweed samples were ground to flour with a miniblender, and then dried with a dryer at 50 °C. The 100 g dried seaweed was treated with 5 L of distilled water and boiled at 100 °C for 30 min, and the extract was centrifuged at 10,000× g for 20 min. The supernatant was added with 4 M CaCl₂ incubated for 1 h to separate alginic acid, and re-centrifuged at 10,000× g for 20 min. All polysaccharides were dialyzed (cut off 10,000 Da) using deionized water for 48 h, and precipitated by the addition of ethanol at the ratio of 1:3 (V/V) and left overnight to give the crude fucoidan. The fucoidan so obtained was fractionated by anion-exchange chromatography using DEAE-Sephadex A-25 (Cl⁻ form, Pharmacia, Uppsala, Sweden) using gradually increasing concentrations of sodium chloride (0–2.5 M) at a flow rate of 1 mL/min, and eluents (5 mL/tube) were separately collected. Each fraction was analyzed for sulfate content (Section 3.3) and monosaccharide composition (Section 3.4). The fraction of rich sulfate and fucose content (fraction 3) was extracted and hydrolyzed with a mixture of crude glycolytic enzymes, isolated from Bacillus subtilis by our lab (Section 3.5). The reaction mixture was passed through a filter (3000 Da cut off) by using Amicon Stirred Cells (Merck Germany) to collect the LMW fucoidan with a molecular weight lower than 3000 Da. The hydrolysis reaction of the fucoidan by the crude glycolytic enzymes was performed at pH 6.5, 37 °C for 24 h and heated at 95 °C for 20 min for inactivation of the enzyme.

The chemicals used for graphene oxide synthesis were the following: natural graphite (Sigma Aldrich, St. Louis, Mo, USA), sulfuric acid (H₂SO₄, 95–97%, Merck, Darmstadt, Germany), potassium persulfate (K₂S₂O₈, Merck, Darmstadt, Germany), phosphorus (V) oxide (P₂O₅, Merck, Darmstadt, Germany), sodium nitrate (NaNO₃, Honeywell and Sigma-Aldrich, Gurgaon, India), potassium permanganate (KMnO₄, Merck, Darmstadt, Germany), hydrogen peroxide (H₂O₂, Merck, Darmstadt, Germany), hydrochloric acid (HCl, 36~38%, Merck, Darmstadt, Germany) and double-distilled water. TiO₂ P25 nanoparticles from Degussa and SiO₂ Aerosil 200 from Evonik (Essen, Germany) were used for the preparation of mixed TiO₂-SiO₂ powder. Acetylacetone and ethanol as organic dispersing agents were purchased from Merck, Darmstadt, Germany and Fluka. Organosilicon compounds namely, octamethylcyclotetrasiloxane, D4 and decamethylcyclopentasiloxane and D5 and biodegradable ethyl-lactate (Et-L) solvent were purchased from Alfa Aesar, Kandel, Germany. Alginic acid sodium salt (NaAlg) and gum rosin (GRos) were obtained from Aldrich, Norwich, United Kingdom and Aveiro, Portugal. Calcium chloride (CaCl₂, Alfa Aesar, Kandel, Germany) was used to promote physical crosslinking and form water-insoluble calcium alginate (CaAlg) on cotton and leather surface.
Bleached 100% cotton woven fabric with the weight of 168 g/m² was used for all experiments. The sheepskin leather surfaces were finished according to the classical technologies by spraying a base coat of acrylic resins with water-based casein pigment and a topcoat of water-based nitrocellulose emulsion [25 (link)]. The cotton and leather materials were provided by the National Research Institute for Leather and Textiles, Bucharest, Romania.

First, a 4% sodium alginate solution was prepared from distilled water and alginic acid sodium salt. It was used throughout the experiment for emulsion preparation as the shell material. Emulsion with Trifolium pratense L. and Glycyrrhiza glabra L. extracts, and Myristica fragrans Houtt. essential oil were prepared as follows: solution with excipients (maltodextrin, inulin, and/ or gum Arabic) was mixed with sodium alginate solution (stirred for 15 min with a magnetic stirrer MSH-20A (Witeg, Wertheim, Germany)) and then extracts with essential oil were added. The solution was homogenised for 15 min at 5000 rpm using an IKA T18 homogeniser (IKA-Werke GmbH & Co., KG, Staufen, Germany).
The emulsion’s stability was tested using a centrifuge Sigma 3-18KS (Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany). The test was repeated three times using 23 °C temperature, 3000 rpm, and the duration was 5 min. The centrifugation index (CI) was calculated to evaluate emulsion stability.
$$\mathrm{CI}(\%)=\frac{{\mathrm{V}}_{\mathrm{e}}}{{\mathrm{V}}_{\mathrm{i}}}·100$$
where V_e is the volume of the remaining emulsion after centrifugation and V_i is the volume of the initial emulsion.

Five stocks of edible coating solutions and their emulsions with essential oils were produced. chitosan solution was made at a final concentration of 1% by dissolving 1 g of chitosan of medium molecular weight obtained from crab shells (48165, Sigma Aldrich, St. Louis, MO, USA) in 100 mL of 1% glacial acetic acid (100056, Merck KGaA, Darmstadt, Germany) and stirring overnight at room temperature. Alginate solution was produced at a final concentration of 1.5% by dissolving 1.5 g of sodium alginate obtained from brown algae (alginic acid sodium salt BioChemica, A3249, AppliChem GmBh, Darmstadt, Germany) in 100 mL of deionized water and stirring overnight at room temperature. chitosan and alginate emulsions with oregano oil were made at a final concentration of 0.1% by dissolving 0.1 mL of oregano oil (W282812, Sigma Aldrich, Burlington, MA, USA) in 100 mL of the stocks of chitosan or alginate solutions and mixing until fully incorporated at room temperature. Alginate emulsion with olive oil was produced at a final concentration of 2% by dissolving 2 mL of olive oil (of extra virgin grade, procured bottled from a local convenience store) in 100 mL of the stock of alginate solution and mixing until well combined at room temperature. All stocks were finally sterilized at 121 °C for 15 min.