I SOLATION AND P URIFICATION

5.3.3 I SOLATION AND P URIFICATION

Two main procedures for polysaccharide isolation from microorganisms exist, depending on whether the biopolymers are part of the cell wall, or excreted by the cells as a capsule or slime (exopolysaccharides). In the former case, isolation is based on extraction from cell mass, while in the latter, centrifugation or filtration is applied to separate product from cells.

For instance, the isolation of cell wall β-D-glucans from S. cerevisiae follows the subsequent steps. 163 After harvesting, the fermentation medium is centrifuged and yeast cells are separated in the sediment, which is then extracted with 6% (w/v) NaOH at 60°C. Polysaccharides, oligosaccharides, and sugars are extracted and subsequently dia- lyzed in water (30 min of stirring). Thus, soluble and low MW substances are removed with water and the remaining fraction (polysaccharides) is centrifuged and the sediment suspended in 3% (w/v) NaOH at 90°C for 2 h. A second round of centrifugation and sediment dialysis against water follows to further purify the

glucan. Finally, the purified glucan is neutralized and extracted with 4% (v/v) H 3 PO 4 (18°C, 2 h). The wet purified glucan is then lyophilized, air dried, or spray dried to form the final product (powder).

A similar but simpler method for isolation of cell wall mannans from S. cere- visiae , Candida albicans, and Candida utilis is described elsewhere 164 and includes extraction of cell debris with 2% (w/v) KOH, removal of impurities from the extract with Fehling’s reagent, and purification of mannans. Isolation of mushroom polysac- charides follows similar procedures, but the cells (fruit bodies) are homogenized or milled before extraction. Specifically, lentinan was isolated after extraction of the homogenized and lyophilized sample with hot (boiling) water for 10 h, subsequent filtration of the suspension to remove insoluble matter, and ethanol precipitation of

Microbial Polysaccharides

the water-soluble polysaccharide, which was then centrifuged and lyophilized. 126 A second extraction cycle after filtration could increase the degree of purity of lentinan.

Extraction protocols of other bioactives from commercial medicinal mushrooms (e.g., antioxidants from Ganoderma lucidum, Ganoderma tsugae, and Coriolus versicolor ) have also been described. 165

For the isolation of several polysaccharides from the same microorganism, a stepwise extraction of the different fractions is adopted, and chromatographic tech- niques can be employed for improved purification. For example, hot-water-soluble polysaccharide extracts from fruit bodies of Agaricus blazei were fractionated and purified after ethanol precipitation, ion exchange chromatography, gel filtration, and affinity chromatography. In this way, four different antitumor biopolymers were

separated. 43 The water-insoluble heteroglycans were obtained by successive extrac- tion with 1% (w/v) ammonium oxalate, 5% (w/v) sodium hydroxide, 20% (w/v) sodium hydroxide, and 5% (w/v) lithium chloride–dimethylacetamide, yielding four polysaccharide fractions. These were precipitated with ethanol and purified chro- matographically. 166

Isolation of extracellular polysaccharides is usually easier and faster, since the main step of filtration or centrifugation (to remove cells) is shorter than extraction

procedures. The speed of centrifugation, filter pore size, and duration of centrifuga- tion or filtration depend on the nature and viscosity of the polysaccharide, and on whether EPS is in the form of slime or capsule (the latter is more difficult to separate from cells). Especially for capsular EPSs, which are closely attached to the cell membrane, a sample pretreatment is usually applied before centrifugation or filtra- tion, to facilitate dissociation of EPSs from cells. This may include alkali addition (e.g., NaOH) or heat treatment of the sample (e.g., at boiling temperature). Some- times the sample is preheated in saline solution or a mixture of water and phenol. Sonication of cell suspension or sample autoclaving can also be used as a treatment to release capsular EPSs from cells. 167

For isolation of scleroglucan, samples of the fermentation medium were initially neutralized (process pH was 4.5) with addition of alkali and diluted fourfold with distilled water. After a heating pretreatment (80°C, 30 min), samples were homog- enized in a blender and centrifuged (27,5000 g, 20 min, 10 to 15°C). The polysac- charide was collected from the supernatant and precipitated by the addition of an equal amount of 96% (v/v) ethanol. After precipitation was completed, scleroglucan was recovered by filtration and dried (105°C). 137 Similar techniques are used for isolation of other water-soluble EPSs, but naturally the pretreatment stages (if any), the intensity of centrifugation, and the volume and type of alcohol vary.

In LAB cultures, isolation and purification of EPS are also based on centrif- ugation and alcohol precipitation of dilute samples, but often include several steps

for removal of proteins and impurities, which are especially essential if the cells are grown on dairy whey or milk. Deproteinization of the samples, after removal of cells, is achieved by treatment with trichloroacetic acid, proteinases, or heating. Ion exchange chromatography and high-performance liquid chromatography (HPLC) can be applied to lyophilized polysaccharides for fractionation and further purification. 67,119

Functional Food Carbohydrates

Water-insoluble exopolysaccharides are separated in a somewhat different way. For example, curdlan from Agrobacterium ATCC 31750 (former Alcaligenes faeca- lis ssp. myxogenes) was isolated as follows: Samples of fermentation broth were diluted with water and centrifuged (8000 g, 30 min, 4°C). The pellet containing cells and curdlan was washed with 0.01 N HCl and precipitated again by centrif- ugation. Subsequently, curdlan was solubilized by addition of 0.5 N NaOH for 1

h. Cells were removed by centrifugation (8000 g, 30 min, 4°C), and curdlan present in the supernatant was recovered (as a pellet) after addition of 2.0 N HCl, and finally dried. 168

It has to be noted that after isolation and purification of the polysaccharide, an additional treatment (derivatization) may be applied for the formulation of the final product, which may aim at increasing its solubility or immunomodulating–prophy- lactic activity. For instance, particulate microbial glucans are often solubilized by carboxy(m)ethylation, sulfation, sulfoethylation, phosphation, or chemical oxida- tion. 5,169–171 This is done to increase the activity of therapeutic polysaccharides and to avoid possible side effects of insoluble or hardly soluble glucans after intraveneous or intraperitoneal administration (inflammation, pain, granuloma formation, micro- embolism, hepatosplenomegaly, circulatory collapse). 5,169,170 In some cases, the derivatives are conjugated with other active compounds or drugs to further improve their performance. 171 This solubilization step could also be useful in a food context, where the functional polysaccharides may need to be dissolved in the water phase of the nutraceutical preparation, so that aggregation of the active compound is prevented, or gel formation and thickening effects of the polysaccharide are facili- tated. In addition, ultrasonic treatment can be used to aid the derivatization of the functional polysaccharides. Also, ultrasounds have been applied for targeted depo- lymerization of polysaccharides to produce lower MW derivatives, which can be more readily dissolved in water, and in some cases are more effective than the original biopolymer. 169 The latter procedure can also be performed by chemical and enzymatic degradation, but ultrasonication is advantageous in that it does not change the chemical nature of the biopolymer, apart from reducing its molecular weight. 169