4.4 Polysaccharides 3354.4.5.3 Cellulases..............................................
Hydrolysis of completely insoluble, microcrys-
talline cellulose is a complicated process. For
this purpose, certain microorganisms produce
particles called cellusomes (particle weight ca.
106 ). During isolation, these particles readily
disintegrate into enzymes, which synergistically
perform cellulose degradation, and components,
which, among other things, support substrate
binding. At least three enzymes are involved in
the degradation of cellulose to cellobiose and
glucose:
(4.168)As shown in Table 4.29, the C 1 and Cxfactors,
which were found to be endo- and exo-1,4-β-
glucanases respectively, hydrolyze cellulose to
cellobiose. Since the C 1 factor is increasingly
inhibited by its product, a cellobiase is needed so
that cellulose breakdown is not rapidly brought to
a standstill. However, cellobiase is also subject to
product inhibition. Therefore, complete cellulose
degradation is possible only if cellobiase is
present in large excess or the glucose formed is
quickly eliminated.
4.4.5.4 Endo-1,3(4)-β-glucanase
This hydrolase is also called laminarinase and
hydrolyzes 1,3(4)-β-glucans. This enzyme occurs
together with cellulases, e. g., in barley malt,
and is involved in the degradation ofβ-glucans
(cf. 15.2.4.2.2) in the production of beer.
4.4.5.5 Hemicellulases
The degradation of hemicelluloses also proceeds
via endo- and exohydrolases. The substrate speci-
ficity depends on the monosaccharide building
blocks and on the type of binding, e. g., endo-1,4-
β-D-xylanase, endo-1,5-α-L-arabinase. These en-
zymes occur in plants and microorganisms, fre-
quently together with cellulases.
4.4.6 Analysis of Polysaccharides
The identification and quantitative determination
of polysaccharides plays a role in the examination
of thickening agents, balast material etc.4.4.6.1 ThickeningAgents
First, thickening agents must be concentrated.
The process used for this purpose is to be modi-
fied depending on the composition of the food. In
general, thickening agents are extracted from the
defatted sample with hot water. Extracted starch
is digested by enzymatic hydrolysis (α-amylase,
glucoamylase), and proteins are separated by
precipitation (e. g., with sulfosalicylic acid). The
polysaccharides remaining in the solution are
separated with ethanol. An electropherogram of
the polysaccharides dissolved in a borate buffer
provides an initial survey of the thickening agents
present. It is sometimes difficult to identify and,
consequently, differentiate between the added
polysaccharides and those that are endogenously
present in many foods. In simple cases, it is
sufficient if the electropherogram is supported
by structural analysis. Here, the polysaccharides
are permethylated (cf. 4.2.4.7), then subjected to
acid hydroysis, reduced with sodium borohydride
(cf. 4.2.4.1) and converted to partially methylated
alditol acetates by acetylation of the OH-groups
(cf. 4.2.4.6).
These derivatives of the monosaccharide
structural units are then qualitatively and quan-
titatively analyzed by gas chromatography
on capillary columns. In more difficult cases,
a preliminary separation of acidic and neu-
tral polysaccharides on an ion exchanger is
recommended. Methanolysis or hydrolysis of
polysaccharides containing uronic acids and
anhydro sugars are critical due to losses of these
labile building blocks.
Reductive cleavage of the permethylated polysac-
charide is recommended as a gentle alternative to
hydrolysis. In this process, partially methylated
anhydroalditolacetates are formed as shown
in Fig. 4.42, using a galactomannan as an
example. Conclusions about the structure of the
polysaccharide can be drawn from the result of
the qualitative and quantitative analysis, which
is carried out by gas chromatography/mass