Analysis of polysaccharides and fiber
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Analysis of starch |
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Starch is the most common digestible polysaccharide found in
foods, and is therefore a major source of energy in our diets. In its natural
form starch exists as water-insoluble granules (3 - 60 mm), but in many
processed foods the starch is no longer in this form because of the processing
treatments involved (e.g., heating). It consists of a mixture of two glucose
homopolysaccharides: amylose (500-2000 glucose units) which is linear, and
amylopectin (>1,000,000 glucose units) which is extensively branched. These
two kinds of starch have different physiochemical properties and so it is often
important to determine the concentration of each individual component of the
starch, as well as the overall starch concentration.
Sample preparation
The starch content of most foods cannot be determined directly
because the starch is contained within a structurally and chemically complex
food matrix. In particular, starch is often present in a semi-crystalline form
(granular or retrograded starch) that is inaccessible to the chemical reagents
used to determine its concentration. It is therefore necessary to isolate
starch from the other components present in the food matrix prior to carrying
out a starch analysis.
In natural foods, such as legumes, cereals or
tubers, the starch granules are usually separated from the other major
components by drying, grinding, steeping in water, filtration and
centrifugation. The starch granules are water-insoluble and have a relatively
high density (1500 kg/m3) so that they will tend to move to the bottom of a
container during centrifugation, where they can be separated from the other
water-soluble and less dense materials. Processed food samples are normally
dried, ground and then dispersed in hot 80% ethanol solutions. The
monosaccharides and oligosaccharides are soluble in the ethanol solution, while
the starch is insoluble. Hence, the starch can be separated from the sugars by
filtering or centrifuging the solution. If any semi-crystalline starch is
present, the sample can be dispersed in water and heated to a temperature where
the starch gelatinizes (> 65oC). Addition of perchloric acid or
calcium chloride to the water prior to heating facilitates the solubilization
of starches that are difficult to extract.
Analysis methods
Once the starch has been extracted there are a number of ways to
determine its concentration:
- Specific enzymes are added to the starch solution to
breakdown the starch to glucose. The glucose concentration is then analyzed
using methods described previously (e.g., chromatography or enzymatic methods). The starch concentration is
calculated from the glucose concentration.
- Iodine can be added to the starch solution to form an
insoluble starch-iodine complex that can be determined
gravimetrically by collecting, drying and
weighing the precipitate formed or titrimetrically by determining the amount of
iodine required to precipitate the starch.
- If there are no other components present in the solution that
would interfere with the analysis, then the starch concentration could be
determined using physical methods, e.g., density, refractive
index or polarimetry.
The amylose and amylopectin concentrations in a sample can be
determined using the same methods as described for starch once the amylose has
been separated from the amylopectin. This can be achieved by adding chemicals
that form an insoluble complex with one of the components, but not with the
other, e.g. some alcohols precipitate amylose but not amylopectin. Some of the
methods mentioned will not determine the concentration of resistant starch
present in the sample. If the concentration of resistant starch is required
then an additional step can be added to the procedure where dimethylsulfoxide
(DMSO) is added to dissolve the resistant starch prior to carrying out the
analysis.
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Analysis of fiber |
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Major components
The major components of dietary fiber are cellulose,
hemicellulose, pectin, hydrocolloids and lignin. Some types of starch, known as
resistant starch, are also indigestible by human beings and may be analyzed as
dietary fiber. The basis of many fiber analysis techniques is therefore to
develop a procedure that mimics the processes that occur in the human digestive
system.
- Cell Wall Polysaccharides: Cellulose occurs in
all plants as the principal structural component of the cell walls, and is
usually associated with various hemicelluloses and lignin. The type and extent
of these associations determines the characteristic textural properties of many
edible plant materials. Cellulose is a long linear homopolysaccahride of
glucose, typically having up to 10,000 glucose subunits. Cellulose molecules
aggregate to form microfibrils that provide strength and rigidity in plant cell
walls. Hemicelluloses are a heterogeneous group of branched
heteropolysaccharides that contain a number of different sugars in their
backbone and side-chains. By definition hemicelluloses are soluble in dilute
alkali solutions, but insoluble in water. Pectins are another form of
heteropolysaccharides found in cell walls that are rich in uronic acids,
soluble in hot water and that are capable of forming gels.
- Non Cell Wall Polysaccharides: This group of
substances are also indigestible carbohydrates, but they are not derived from
the cell walls of plants. Non-cell wall polysaccharides include hydrocolloids
such as guar and locust bean gum, gum arabic, agar, alginates and caragenans
which are commonly used in foods as gelling agents, stabilizers and thickeners.
- Lignin:Lignin is a non-carbohydrate polymer that
consists of about 40 aromatic subunits which are covalently linked. It is
usually associated with cellulose and hemicelluloses in plant cell-walls.
There are a number of procedures that are commonly used in many
of the methods for dietary fiber analysis:
- Lipid removal. The food sample to be analyzed is
therefore dried, ground to a fine powder and then the lipids are removed by
solvent extraction.
- Protein removal. Proteins are usually broken down and
solubilized using enzymes, strong acid or strong alkali solutions. The
resulting amino acids are then separated from insoluble fiber by filtration or
from total fiber by selective precipitation of the fiber with ethanol
solutions.
- Starch removal. Semi-crystalline starch is gelatinized
by heating in the presence of water, and then the starch is broken down and
solubilized by specific enzymes, strong acid or strong alkali. The glucose is
then separated from insoluble fiber by filtration or separated from total fiber
by selective precipitation of the fiber with ethanol solutions.
- Selective precipitation of fibers. Dietary fibers can
be separated from other components in aqueous solutions by adding different
concentrations of ethanol to cause selective precipitation. The solubility of
monosaccharides, oligosaccharides and polysaccharides depends on the ethanol
concentration. Water: monosaccharides, oligosaccharides, some
polysaccharides and amino acids are soluble; other polysaccharides and fiber
are insoluble. 80% ethanol solutions: monosaccharides, oligosaccharides
and amino acids are soluble; polysaccharides and fibers are insoluble. For this
reason, concentrated ethanol solutions are often used to selectively
precipitate fibers from other components.
- Fiber analysis. The fiber content of a food can
be determined either gravimetrically by weighing the mass of an
insoluble fiber fraction isolated from a sample or chemically by
breaking down the fiber into its constituent monosaccharides and measuring
their concentration using the methods described previously.
Gravimetric methods
The crude fiber method gives an estimate of indigestible fiber
in foods. It is determined by sequential extraction of a defatted sample or by
selective precipitation after enzymatic solubilization. for deatils: Proximate
analysis < Analysis of crude fiber
Chemical Methods: the Englyst-Cummings Procedure
In this chemical methods, the fiber content is equal to the sum
of all non-starch monosaccharides plus lignin remaining once all the digestible
carbohydrates have been removed. Monosaccharides are measured using the various
methods described previously.
A defatted food sample is heated in water to gelatinize the
starch. Enzymes are then added to digest the starch and proteins. Pure ethanol
is added to the solution to precipitate the fiber, which is separated from the
digest by centrifugation, and is then washed and dried. The fiber is then
hydrolyzed using a concentrated sulfuric acid solution to break it down into
its constituent monosaccharides, whose concentration is determined using the
methods described previously, e.g., colorimetrically or chromatographically.
The mass of fiber in the original sample is assumed to be equal to the total
mass of monosaccharides present. The concentration of insoluble and soluble
dietary fiber can also be determined by this method, using similar separation
steps as for the total, insoluble and soluble gravimetric method mentioned
above.
This method can be used to determine the total, soluble and
insoluble fiber contents of foods, but does not provide information about the
lignin content. This is because lignin is not a polysaccharide, and so it is
not broken down to monosaccharides during the acid digestion. For most foods
this is not a problem because they have low lignin concentrations anyway. If a
food does contain significant amounts of lignin then another method should be
used, e.g., the gravimetric method or more sophisticated chemical methods
(e.g., the Theander-Marlett method).
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