7. Analysis of Carbohydrates

7.1 Introduction

Carbohydrates are one of the most important components in many foods. Carbohydrates may be present as isolated molecules or they may be physically associated or chemically bound to other molecules. Individual molecules can be classified according to the number of monomers that they contain as monosaccharides, oligosaccharides or polysaccharides. Molecules in which the carbohydrates are covalently attached to proteins are known as glycoproteins, whereas those in which the carbohydrates are covalently attached to lipids are known as glycolipids. Some carbohydrates are digestible by humans and therefore provide an important source of energy, whereas others are indigestible and therefore do not provide energy. Indigestible carbohydrates form part of a group of substances known as dietary fiber, which also includes lignin. Consumption of significant quantities of dietary fiber has been shown to be beneficial to human nutrition, helping reduce the risk of certain types of cancer, coronary heart disease, diabetes and constipation. As well as being an important source of energy and dietary fiber, carbohydrates also contribure to the sweetness, appearence and textural characteristics of many foods. It is important to determine the type and concentration of carbohydrates in foods for a number of reasons.

 

7.2. Classification of Carbohydrates

Monosaccharides

Monosaccharides are water-soluble crystalline compounds. They are aliphatic aldehydes or ketones which contain one carbonyl group and one or more hydroxyl groups. Most natural monosachharides have either five (pentoses) or six (hexoses) carbon atoms. Commonly occurring hexoses in foods are glucose, fructose and galactose, whilst commonly occurring pentoses are arabinose and xylose. The reactive centers of monosaccharides are the carbonyl and hydroxyl groups.

Oligosaccharides

These are relatively low molecular weight polymers of monosaccharides (< 20) that are covalently bonded through glycosidic linkages. Disaccharides consist of two monomers, whereas trisaccharides consist of three. Oligosaccharides containing glucose, fructose and galactose monomers are the most commonly occurring in foods.

Polysaccharides

The majority of carbohydrates found in nature are present as polysaccharides. Polysaccharides are high molecular weight polymers of monosaccharides (> 20). Polysaccharides containing all the same monosaccharides are called homopolysaccharides (e.g., starch, cellulose and glycogen are formed from only glucose), whereas those which contain more than one type of monomer are known as heteropolysaccharides (e.g., pectin, hemicellulose and gums).

 

7.3. Methods of Analysis

A large number of analytical techniques have been developed to measure the total concentration and type of carbohydrates present in foods (see Food Analysis by Nielssen or Food Analysis by Pomeranz and Meloan for more details). The carbohydrate content of a food can be determined by calculating the percent remaining after all the other components have been measured: %carbohydrates = 100 - %moisture - %protein - %lipid - %mineral. Nevertheless, this method can lead to erroneous results due to experimental errors in any of the other methods, and so it is usually better to directly measure the carbohydrate content for accurate measurements.

 

7.4. Monosaccharides and Oligosaccharides

7.4.1. Sample Preparation

The amount of preparation needed to prepare a sample for carbohydrate analysis depends on the nature of the food being analyzed. Aqueous solutions, such as fruit juices, syrups and honey, usually require very little preparation prior to analysis. On the other hand, many foods contain carbohydrates that are physically associated or chemically bound to other components, e.g., nuts, cereals, fruit, breads and vegetables. In these foods it is usually necessary to isolate the carbohydrate from the rest of the food before it can be analyzed. The precise method of carbohydrate isolation depends on the carbohydrate type, the food matrix type and the purpose of analysis, however, there are some procedures that are common to many isolation techniques. For example, foods are usually dried under vacuum (to prevent thermal degradation), ground to a fine powder (to enhance solvent extraction) and then defatted by solvent extraction.

One of the most commonly used methods of extracting low molecular weight carbohydrates from foods is to boil a defatted sample with an 80% alcohol solution. Monosaccharides and oligosaccharides are soluble in alcoholic solutions, whereas proteins, polysaccharides and dietary fiber are insoluble. The soluble components can be separated from the insoluble components by filtering the boiled solution and collecting the filtrate (the part which passes through the filter) and the retentante (the part retained by the filter). These two fractions can then be dried and weighed to determine their concentrations. In addition, to monosaccharides and oligosaccharides various other small molecules may also be present in the alcoholic extract that could interfere with the subsequent analysis e.g., amino acids, organic acids, pigments, vitamins, minerals etc. It is usually necessary to remove these components prior to carrying out a carbohydrate analysis. This is commonly achieved by treating the solution with clarifying agents or by passing it through one or more ion-exchange resins.

Prior to analysis, the alcohol can be removed from the solutions by evaporation under vacuum so that an aqueous solution of sugars remains.

7.4.2. Chromatographic and Electrophoretic methods

Chromatographic methods are the most powerful analytical techniques for the analysis of the type and concentration of monosaccharides and oligosaccharides in foods. Thin layer chromatography (TLC), Gas chromatography (GC) and High Performance Liquid chromatography (HPLC) are commonly used to separate and identify carbohydrates. Carbohydrates are separated on the basis of their differential adsorption characteristics by passing the solution to be analyzed through a column. Carbohydrates can be separated on the basis of their partition coefficients, polarities or sizes, depending on the type of column used. HPLC is currently the most important chromatographic method for analyzing carbohydrates because it is capable of rapid, specific, sensitive and precise measurements. In addition, GC requires that the samples be volatile, which usually requires that they be derivitized, whereas in HPLC samples can often be analyzed directly. HPLC and GC are commonly used in conjunction with NMR or mass spectrometry so that the chemical structure of the molecules that make up the peaks can also be identified.

Carbohydrates can also be separated by electrophoresis after they have been derivitized to make them electrically charged, e.g., by reaction with borates. A solution of the derivitized carbohydrates is applied to a gel and then a voltage is applied across it. The carbohydrates are then separated on the basis of their size: the smaller the size of a carbohydrate molecule, the faster it moves in an electrical field.

7.4.3. Chemical methods

A number of chemical methods used to determine monosaccharides and oligosaccharides are based on the fact that many of these substances are reducing agents that can react with other components to yield precipitates or colored complexes which can be quantified. The concentration of carbohydrate can be determined gravimetrically, spectrophotometrically or by titration. Non-reducing carbohydrates can be determined using the same methods if they are first hydrolyzed to make them reducing. It is possible to determine the concentration of both non-reducing and reducing sugars by carrying out an analysis for reducing sugars before and after hydrolyzation. Many different chemical methods are available for quantifying carbohydrates. Most of these can be divided into three catagories: titration, gravimetric and colorimetric. An example of each of these different types is given below.

Titration Methods

The Lane-Eynon method is an example of a tritration method of determining the concentration of reducing sugars in a sample. A burette is used to add the carbohydrate solution being analyzed to a flask containing a known amount of boiling copper sulfate solution and a methylene blue indicator. The reducing sugars in the carbohydrate solution react with the copper sulfate present in the flask. Once all the copper sulfate in solution has reacted, any further addition of reducing sugars causes the indicator to change from blue to white. The volume of sugar solution required to reach the end point is recorded. The reaction is not stoichemetric, which means that it is necessary to prepare a calibration curve by carrying out the experiment with a series of standard solutions of known carbohydrate concentration.

The disadvantages of this method are (i) the results depend on the precise reaction times, temperatures and reagent concentrations used and so these parameters must be carefully controlled; (ii) it cannot distinguish between different types of reducing sugar, and (iii) it cannot directly determine the concentration of non-reducing sugars, (iv) it is sucseptible to interference from other types of molecules that act as reducing agents..

Gravimetric Methods

The Munson and Walker method is an example of a gravimetric method of determining the concentration of reducing sugars in a sample. Carbohydrates are oxidized in the presence of heat and an excess of copper sulfate and alkaline tartrate under carefully controlled conditions which leads to the formation of a copper oxide precipitate:

reducing sugar + Cu2+ + base oxidized sugar + CuO2 (precipitate)

The amount of precipitate formed is directly related to the concentration of reducing sugars in the initial sample. The concentration of precipitate present can be determined gravimetrically (by filtration, drying and weighing), or titrimetrically (by redissolving the precipitate and titrating with a suitable indicator). This method suffers from the same disadvantages as the Lane-Eynon method, neverthless, it is more reproducible and accurate.

Colorimetric Methods

The Anthrone method is an example of a colorimetric method of determining the concentration of the total sugars in a sample. Sugars react with the anthrone reagent under acidic conditions to yield a blue-green color. The sample is mixed with sulfuric acid and the anthrone reagent and then boiled until the reaction is completed. The solution is then allowed to cool and its absorbance is measured at 620 nm. There is a linear relationship between the absorbance and the amount of sugar that was present in the original sample. This method determines both reducing and non-reducing sugars because of the presence of the strongly oxidizing sulfuric acid. Like the other methods it is non-stoichemetric and therefore it is necessary to prepare a calibration curve using a series of standards of known carbohydrate concentration.

The Phenol - Sulfuric Acid method is an example of a colorimetric method that is widely used to determine the total concentration of carbohydrates present in foods. A clear aqueous solution of the carbohydrates to be analyzed is placed in a test-tube, then phenol and sulfuric acid are added. The solution turns a yellow-orange color as a result of the interaction between the carbohydrates and the phenol. The absorbance at 420 nm is proportional to the carbohydrate concentration initially in the sample. The sulfuric acid causes all non-reducing sugars to be converted to reducing sugars, so that this method determines the total sugars present. This method is non-stoichemetric and so it is necessary to prepare a calibration curve using a series of standards of known 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 the previous method. The maltose and sucrose are broken down into their constituent monosaccharides by the enzyme a-glucosidase:

maltose + H2O 2 glucose

sucrose +H2O glucose + fructose

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 a-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 a = [a]lc, where a is the measured angle of rotation, [a] 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 a versus concentration is prepared using a series of solutions with known concentration, or the value of [a] is 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/cm). The refractive index of a 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 (Snells Law: sin(i)/sin(r) = n2/n1). In practice, the refractive index of carbohydrate 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 wavelength dependent and so measurements are usually made at a specific temperature (20 oC) and wavelength (589.3nm). This method is quick and simple to carry out and can be performed with simple hand-held instruments. It is used routinely in industry to determine sugar concentrations of syrups, honey, molasses, tomato products and jams.

Density

The density of a material is its mass divided by its volume. The density of aqueous solutions increases as the carbohydrate concentration increases. Thus the carbohydrate concentration can be determined by measuring density, e.g., using density bottles or hydrometers. This technique is routinely used in industry for determination of carbohydrate concentrations of juices and beverages.

Infrared

A material absorbs infrared due to vibration or rotation of molecular groups. Carbohydrates contain molecular groups that absorb infrared radiation at wavelengths where none of the other major food constituents absorb consequently their concentration can be determined by measuring the infrared absorbance at these wavelengths. By carrying out measurements at a number of different specific wavelengths it is possible to simultaneously determine the concentration of carbohydrates, proteins, moisture and lipids. Measurements are normally carried out by measuring the intensity of an infrared wave reflected from the surface of a sample: the greater the absorbance, the lower the reflectance. Analytical instruments based on infrared absorbance are non-destructive and capable of rapid measurements and are therefore particularly suitable for on-line analysis or for use in a quality control laboratory where many samples are analyzed routinely.

More sophisticated instrumental methods are capable of providing information about the molecular structure of carbohydrates as well as their concentration, e.g., NMR or mass spectrometry.

7.4.6. Immunoassays

Immuoassays are finding increasing use in the food industry for the qualitative and quantitative analysis of food products. Immunoassays specific for low molecular weight carbohydrates are developed by attaching the carbohydrate of interest to a protein, and then injecting it into an animal. With time the animal develops antibodies specific for the carbohydrate molecule. These antibodies can then be extracted from the animal and used as part of a test kit for determining the concentration of the specific carbohydrate in foods. Immuoassays are extremely sensitive, specific, easy to use and rapid.

 

7.5 Analysis of Polysaccharides and Fiber

A wide variety of polysaccharides occur in foods. Polysaccharides can be classified according to their molecular characteristics (e.g., type, number, bonding and sequence of monosaccharides), physicochemical characteristics (e.g., water solubility, viscosity, surface activity) and nutritional function (e.g., digestible or non-digestible). Most polysaccharides contain somewhere between 100 and several thousand monosaccharides. Some polysaccharides contain all the same kind of monosaccharide (homopolysaccharides), whereas others contain a mixture of different kinds of monosaccharide (heteropolysaccharides). Some polysaccharides exist as linear chains, whereas others exist as branched chains. Some polysaccharides can be digested by human beings and therefore form an important source of energy (e.g., starch), whereas others are indigestible (e.g., cellulose, hemicellulose and pectins). These indigestible polysaccharides form part of a group of substances known as dietary fiber, which also includes lignin (which is a polymer of aromatic molecules). Consumption of many types of dietary fiber has been shown to have beneficial physiologically functional properties for humans, e.g., prevention of cancer, heart disease and diabetes.

7.5.1. Analysis of Starch

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 (> 65 oC). 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:

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.

 

7.5.2. Analysis of Fibers

Over the past twenty years or so nutritionists have become aware of the importance of fiber in the diet. Liberal consumption of fiber helps protect against colon cancer, cardiovascular disease and constipation. Adequate intake of dietary fiber is therefore beneficial to good health. Dietary fiber is defined as plant polysaccharides that are indigestible by humans, plus lignin. 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.

7.5.2.1. Major Components of Dietary Fiber

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.

7.5.2.2. Common Procedures in Sample Preparation and Analysis

There are a number of procedures that are commonly used in many of the methods for dietary fiber analysis:

 

7.5.2.3. Gravimetric Methods

Crude Fiber Method

The crude fiber method gives an estimate of indigestible fiber in foods. It is determined by sequential extraction of a defatted sample with 1.25% H2SO4 and 1.25% NaOH. The insoluble residue is collected by filtration, dried, weighed and ashed to correct for mineral contamination of the fiber residue. Crude fiber measures cellulose and lignin in the sample, but does not determine hemicelluloses, pectins and hydrocolloids, because they are digested by the alkali and acid and are therefore not collected. For this reason many food scientists believe that its use should be discontinued. Nevertheless, it is a fairly simple method to carry out and is the official AOAC method for a number of different foodstuffs.

Total, insoluble and soluble fiber method

The basic principle of this method is to isolate the fraction of interest by selective precipitation and then to determine its mass by weighing. A gelatinized sample of dry, defatted food is enzymatically digested with a-amylase, amyloglucosidase and protease to break down the starch and protein components. The total fiber content of the sample is determined by adding 95% ethanol to the solution to precipitate all the fiber. The solution is then filtered and the fiber is collected, dried and weighed. Alternatively, the water-soluble and water-insoluble fiber components can be determined by filtering the enzymatically digested sample. This leaves the soluble fiber in the filtrate solution, and the insoluble fiber trapped in the filter. The insoluble component is collected from the filter, dried and weighed. The soluble component is precipitated from solution by adding 95% alcohol to the filtrate, and is then collected by filtration, dried and weighed. The protein and ash content of the various fractions are determined so as to correct for any of these substances which might remain in the fiber: Fiber = residue weight - weight of (protein + ash).

This method has been officially sanctioned by the AOAC and is widely used in the food industry to determine the fiber content of a variety of foods. Its main disadvantage is that it tends to overestimate the fiber content of foods containing high concentrations of simple sugars, e.g., dried fruits, possibly because they get trapped in the precipitates formed when the ethanol is added.

 

7.5.2.4. Chemical Methods

In chemical methods, the fiber content is equal to the sum of all nonstarch monosaccharides plus lignin remaining once all the digestible carbohydrates have been removed. Monosaccharides are measured using the various methods described previously.

Englyst-Cummings Procedure

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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