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carbohydrate concentration.
7.4.4. Enzymatic Methods
Analytical methods based on enzymes rely on their ability to catalyze specific
reactions. These methods are rapid, highly specific and sensitive to low concentrations
and are therefore ideal for determination of carbohydrates in foods. In addition, little
sample preparation is usually required. Liquid foods can be tested directly, whereas
solid foods have to be dissolved in water first. There are many enzyme assay kits
which can be purchased commercially to carry out analysis for specific carbohydrates.
Manufacturers of these kits provide detailed instructions on how to carry out the
analysis. The two methods most commonly used to determine carbohydrate
concentration are: (i) allowing the reaction to go to completion and measuring the
concentration of the product, which is proportional to the concentration of the initial
substrate; (ii). measuring the initial rate of the enzyme catalyzed reaction because the
rate is proportional to the substrate concentration. Some examples of the use of
enzyme methods to determine sugar concentrations in foods are given below:
D-Glucose/D-Fructose
This method uses a series of steps to determine the concentration of both
glucose and fructose in a sample. First, glucose is converted to glucose-6-phosphate
(G6P) by the enzyme hexakinase and ATP. Then, G6P is oxidized by NADP+ in the
presence of G6P-dehydrogenase (G6P-DH)
G6P + NADP+ ’! gluconate-6-phosphate + NADPH + H+
The amount of NADPH formed is proportional to the concentration of G6P in
the sample and can be measured spectrophotometrically at 340nm. The fructose
concentration is then determined by converting the fructose into glucose, using another
specific enzyme, and repeating the above procedure.
Maltose/Sucrose
The concentration of maltose and sucrose (disaccharides) in a sample can be
determined after the concentration of glucose and fructose have been determined by
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the previous method. The maltose and sucrose are broken down into their constituent
monosaccharides by the enzyme ±-glucosidase:
maltose + H O ’! 2 glucose
2
sucrose +H O ’! glucose + fructose
2
The concentrations of glucose and fructose can then be determined by the
previous method. The major problem with this method is that many other
oligosaccharides are also converted to monosaccharides by ± -glucosidase, and it is
difficult to determine precisely which oligosaccharides are present. This method is
therefore useful only when one knows the type of carbohydrates present, but not their
relative concentrations. Various other enzymatic methods are available for determining
the concentration of other monosaccharides and oligosaccharides, e.g., lactose,
galactose and raffinose (see Food Analysis Nielssen).
7.4.5. Physical Methods
Many different physical methods have been used to determine the
carbohydrate concentration of foods. These methods rely on their being a change in
some physicochemical characteristic of a food as its carbohydrate concentration varies.
Commonly used methods include polarimetry, refractive index, IR, and density.
Polarimetry
Molecules that contain an asymmetric carbon atom have the ability to rotate
plane polarized light. A polarimeter is a device that measures the angle that plane
polarized light is rotated on passing through a solution. A polarimeter consists of a
source of monochromatic light, a polarizer, a sample cell of known length, and an
analyzer to measure the angle of rotation. The extent of polarization is related to the
concentration of the optically active molecules in solution by the equation
± = [ ± ]lc, where ± is the measured angle of rotation, [± ] is the optical activity
(which is a constant for each type of molecule), l is the pathlength and c is the
concentration. The overall angle of rotation depends on the temperature and
wavelength of light used and so these parameters are usually standardized to 20oC and
589.3 nm (the D-line for sodium). A calibration curve of ± versus concentration is
prepared using a series of solutions with known concentration, or the value of [± ] is
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taken from the literature if the type of carbohydrates present is known. The
concentration of carbohydrate in an unknown sample is then determined by measuring
its angle of rotation and comparing it with the calibration curve.
Refractive Index
The refractive index (n) of a material is the velocity of light in a vacuum
divided by the velocity of light in the material (n = c/c ). The refractive index of a
m
material can be determined by measuring the angle of refraction (r) and angle of
incidence (i) at a boundary between it and another material of known refractive index
(Snell s Law: sin(i)/sin(r) = n /n ). In practice, the refractive index of carbohydrate
2 1
solutions is usually measured at a boundary with quartz. The refractive index of a
carbohydrate solution increases with increasing concentration and so can be used to
measure the amount of carbohydrate present. The RI is also temperature and
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