General Properties, Occurrence, and Preparation
1.2
91
approximately one-sixth of a cake of baker’s yeast is finely suspended in 10–15 mL of water
and added to the clear filtrate that is covered with a cotton plug. Fermentation is allowed to
go ≈15 h at 37 °C to remove D-glucose (if crude xylan is used, this step can be omitted).
After removal of the yeast, activated charcoal is added with an equal weight of Celite 535
and the mixture is filtered by suction. The filtrate is concentrated to a syrup under reduced
pressure at 50–60 °C, and three volumes of methanol are added with stirring. The solution
is filtered and concentrated to syrup under reduced pressure. The syrup is dissolved in water
and passed through a column (5.5 × 50 cm) of equal amounts of activated charcoal and Celite
535. The column is then washed with 6 L of water and the washings and the original filtrate
are combined and concentrated under pressure to a syrup (∼30 mL) that is filtered through
a coarse sintered-glass filter. The filtrate is allowed to stand at 20 °C until crystallization of
α-D-xylopyranose is complete. The crystals are removed by filtration and washed with cold
(4 °C) 85% (v/v) aqueous methanol. A second crop of crystals are usually obtained by placing
the supernatant at 4 °C [151].
15.2 Isolation and Purification of Lactose from Milk
Commercial skimmed (defatted) milk contains ∼3% casein, 0.7% albumin, 4–5% lactose, and
1% minerals, along with small amounts of lactosamine and lactosamine oligosaccharides, with
the remainder being water. The casein is first precipitated by warning to 40 °C and the addition
of 1:10 (v/v) glacial acetic acid and water to 200 mL of milk, with continuous stirring. The
dilute acetic acid is added until casein no longer separates. The precipitated casein is removed
by centrifugation. Then 5 g of calcium carbonate is immediately added and stirred for ∼5 min
and then the solution is heated to boiling for ∼10 min. This produces almost complete precipitation of the albumin, which is removed by vacuum filtration. The filtrate is concentrated by
roto-vacuum evaporation to ∼30 mL. Then 166 mL of hot ethanol is added, along with 5 g of
activated charcoal; after it has been mixed well, the warm solution is filtered through a bed
of Celite. The clear filtrate is allowed to stand 15–25 h at 20 °C or longer for crystallization.
When crystallization is complete, the crystals are removed by filtration and a second crop of
crystals are obtained by placing the clear solution at 4 °C.
15.3 Analysis, Isolation, and Purification
of Monosaccharides and Oligosaccharides
Individual monosaccharides and their reduced sugar alcohols can be separated and analyzed
by multiple ascent silica-gel, thin-layer chromatography [152], as well as the more complex
mixtures of a series of homologous oligosaccharides, such as maltodextrins, isomaltodextrans,
cellodextrins, chitosan- and chito-dextrins, cyclomaltodextrins, and the raffinose-sucrose dextrins can be quantitatively analyzed by multiple ascent silica-gel, thin-layer chromatography
(TLC), followed by scanning densitometry [152,153,154]. See > Fig. 8 for a TLC separation
of maltodextrins and cyclomaltodextrins. Pure individual oligosaccharides can be obtained
in 50–200 mg amounts by preparative descending paper chromatography, using 70:30 (v/v)
propanol-1/water solvent on 23 × 54 cm Whatman 3MM paper for 24–36 h on which the saccharides are separated, and detected by AgNO 3 /NaOH/Na 2 S 2 O 3 development of a 1-cm strip
1.2
91
approximately one-sixth of a cake of baker’s yeast is finely suspended in 10–15 mL of water
and added to the clear filtrate that is covered with a cotton plug. Fermentation is allowed to
go ≈15 h at 37 °C to remove D-glucose (if crude xylan is used, this step can be omitted).
After removal of the yeast, activated charcoal is added with an equal weight of Celite 535
and the mixture is filtered by suction. The filtrate is concentrated to a syrup under reduced
pressure at 50–60 °C, and three volumes of methanol are added with stirring. The solution
is filtered and concentrated to syrup under reduced pressure. The syrup is dissolved in water
and passed through a column (5.5 × 50 cm) of equal amounts of activated charcoal and Celite
535. The column is then washed with 6 L of water and the washings and the original filtrate
are combined and concentrated under pressure to a syrup (∼30 mL) that is filtered through
a coarse sintered-glass filter. The filtrate is allowed to stand at 20 °C until crystallization of
α-D-xylopyranose is complete. The crystals are removed by filtration and washed with cold
(4 °C) 85% (v/v) aqueous methanol. A second crop of crystals are usually obtained by placing
the supernatant at 4 °C [151].
15.2 Isolation and Purification of Lactose from Milk
Commercial skimmed (defatted) milk contains ∼3% casein, 0.7% albumin, 4–5% lactose, and
1% minerals, along with small amounts of lactosamine and lactosamine oligosaccharides, with
the remainder being water. The casein is first precipitated by warning to 40 °C and the addition
of 1:10 (v/v) glacial acetic acid and water to 200 mL of milk, with continuous stirring. The
dilute acetic acid is added until casein no longer separates. The precipitated casein is removed
by centrifugation. Then 5 g of calcium carbonate is immediately added and stirred for ∼5 min
and then the solution is heated to boiling for ∼10 min. This produces almost complete precipitation of the albumin, which is removed by vacuum filtration. The filtrate is concentrated by
roto-vacuum evaporation to ∼30 mL. Then 166 mL of hot ethanol is added, along with 5 g of
activated charcoal; after it has been mixed well, the warm solution is filtered through a bed
of Celite. The clear filtrate is allowed to stand 15–25 h at 20 °C or longer for crystallization.
When crystallization is complete, the crystals are removed by filtration and a second crop of
crystals are obtained by placing the clear solution at 4 °C.
15.3 Analysis, Isolation, and Purification
of Monosaccharides and Oligosaccharides
Individual monosaccharides and their reduced sugar alcohols can be separated and analyzed
by multiple ascent silica-gel, thin-layer chromatography [152], as well as the more complex
mixtures of a series of homologous oligosaccharides, such as maltodextrins, isomaltodextrans,
cellodextrins, chitosan- and chito-dextrins, cyclomaltodextrins, and the raffinose-sucrose dextrins can be quantitatively analyzed by multiple ascent silica-gel, thin-layer chromatography
(TLC), followed by scanning densitometry [152,153,154]. See > Fig. 8 for a TLC separation
of maltodextrins and cyclomaltodextrins. Pure individual oligosaccharides can be obtained
in 50–200 mg amounts by preparative descending paper chromatography, using 70:30 (v/v)
propanol-1/water solvent on 23 × 54 cm Whatman 3MM paper for 24–36 h on which the saccharides are separated, and detected by AgNO 3 /NaOH/Na 2 S 2 O 3 development of a 1-cm strip