The preceding sections indicate that the carbohydrate analyst has a wide range of options for the determination of mono- and disaccharides in foods. The current state of chromatographic techniques can provide powerful and versatile analytical methods allowing for both selective and sensitive determination. The approach selected may well be dependent on the chromatographic equipment available in the food analysis laboratory. Gas chromatographic determination
Mono- and Disaccharides: Analytical Aspects 37
requires only a basic instrument and flame ionization detection. For routine high-performance liquid chromatography determination of common food sug-ars, a simple isocratic system incorporating an aminopropyl–silica column and a refractive index detector is adequate. The range of analyses can be extended by the use of a fixed-ion resin column. An alternative detector is the evaporative light-scattering detector, which provides greater sensitivity and the potential for use in more complex separations requiring gradient elution. Where high sensitivity and selectivity are demanded by the analysis, a more sophisticated HPLC system based on anion-exchange chromatography and pulsed ampero-metric detection is likely to be the best solution. The analyst must give serious consideration to all the determinations that may be required before deciding on the analytical strategy and selecting the chromatography system. The actual procedures adopted for optimum quantitative determinations are very dependent on the mono- and disaccharides to be determined and the nature of the samples to be analyzed.
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2 Mono- and Disaccharides: Selected Physicochemical and Functional Aspects
Kirsi Jouppila
CONTENTS
2.1 Introduction...42 2.2 Molecular Structure of Mono- and Disaccharides...43 2.3 Mono- and Disaccharides in Water Solutions...44 2.3.1 Solubility...44 2.3.2 Mutarotation...45 2.3.3 Effect of Mono- and Disaccharides on Colligative Properties .... 46 2.4 Mono- and Disaccharides in Solid Form ...47 2.4.1 Crystalline State and Melting of Sugars ...47 2.4.2 Water Sorption of Crystalline and Amorphous Sugars...49 2.4.3 Glass Transition and Plasticization of Amorphous Sugars...52 2.4.4 Critical Values for Water Content
and Storage Relative Humidity ...57 2.5 Crystallization of Mono- and Disaccharides...60 2.5.1 Crystallization in Solutions ...61 2.5.2 Amorphous State and Crystallization...61 2.5.2.1 Effect of Plasticization on Crystallization ...62 2.5.2.1.1 Gravimetric Studies...62 2.5.2.1.2 XRD Studies ...63 2.5.2.1.3 Thermoanalytical Studies...65 2.5.2.1.4 Other Techniques ...68 2.5.2.2 Kinetics of Crystallization ...68 2.5.2.3 Leveling-Off Extent of Crystallization ...74 2.5.2.4 Crystal Forms ...76 2.6 Summary and Conclusions ...80 References ...82
2.1 INTRODUCTION
Mono- and disaccharides are sugars containing carbon, oxygen, and hydrogen atoms, and they are classified as carbohydrates, which also include oligo- and polysaccharides. Mono- and disaccharides are the lowest molecular weight carbohydrates. They are formed in plants [1], and they can be separated from plant material using, for example, water extraction followed by crystallization [2]. Mono- and disaccharides are often stored in stable crystal form. They are added to foods to increase sweetness, to give color and flavor (as a result of nonenzymatic browning and caramellization reactions), and to increase storage stability by lowering the water activity (aw) of a product. The physicochemical and functional properties (sweetness, solubility, melting temperature, glass transition temperature, and reactivity) of mono- and disaccharides differ, although the molecular structures are quite similar.
Mono- and disaccharides may exist in amorphous form with a random molecular order in some food products, as reviewed by White and Cakebread [3] and Roos [4]. Such products include dehydrated food products, such as milk and whey powders, that are obtained via spray-drying, as well as freeze-dried fruit and berries and hard sugar candies that have been produced by rapid cooling.
Also, other food processes, such as freezing and extrusion cooking, can produce amorphous (noncrystalline), often solid structures, such as a freeze-concentrated, unfrozen phase of frozen products (e.g., in ice cream) and crisp and brittle snack products, respectively. Amorphous sugar and a sugar-containing matrix may encapsulate various compounds, such as aroma compounds and bioactive sub-stances, as reviewed by Karel [5]. Encapsulation may increase the stability of these compounds by preventing oxidation of the encapsulated compounds.
Amorphous food materials are in a glassy, metastable state if they are stored at temperatures lower than their glass transition temperature [3,4,6].
Water plasticizes amorphous food materials, resulting in lowered glass tran-sition temperatures. If the glass trantran-sition temperature is lower than the ambi-ent temperature, molecular mobility in the material increases, and various changes may occur during food processing and storage. Stickiness and caking are desired phenomena in the agglomeration process but are undesired changes during the storage of food powders. Crystallization of sugars is often an undesired phenomenon during storage; for example, crystallization of lactose can decrease the quality of dairy powders because of the poor solubility of crystalline lactose and affects the quality of ice cream because crystallized lactose gives an unpleasant, sandy mouthfeel [3]. Crystallizing, however, is the main unit operation for producing crystalline sugars, so controlling crys-tallization might involve either preventing cryscrys-tallization or promoting crystal formation and growth, as reviewed by Hartel [7,8]. Water sorption and water plasticization data for amorphous sugars and sugar-containing products are necessary to predict the occurrence and rate of various potential changes during processing and storage [4,6].
Mono- and Disaccharides: Physicochemical and Functional Aspects 43
This chapter discusses the selected physicochemical and functional aspects of mono- and disaccharides, with an emphasis on amorphous solid states of sugars and plasticization of amorphous sugars in relation to time-dependent changes, mainly crystallization.
2.2 MOLECULAR STRUCTURE OF MONO- AND DISACCHARIDES
Monosaccharides are the simplest form of sugars with a molecular formula of Cn(H2O)n, where n ranges from 3 to 9 [9,10]. The most common monosaccha-rides are hexoses, such as D-glucose and D-fructose, and pentoses, such as D -arabinose and L-arabinose [1]. Monosaccharides having the same molecular weight but different molecular structures, as well as different chemical and physical properties, are isomers to each other. The two fundamental types of isomers are structural and spatial, both of which occur in monosaccharides [1].
Structural isomers of monosaccharides may be functional group isomers:
They contain either an aldehyde or a ketone group in their molecular structure and are classified as aldoses (e.g., aldohexose) or ketoses (e.g., hexulose), respectively [1,10]. Structural isomers of monosaccharides may exist in ring forms of different sizes: a five-membered furanose (furan-like) ring or a six-membered pyranose (pyran-like) ring resulting from formation of intramolec-ular hemiacetals or hemiketals [9]. The carbon atom of the carbonyl group becomes chiral when it is involved in ring formation, resulting in two different anomeric forms of monosaccharide (e.g., α-D-glucopyranose, β-D -glucopyra-nose). Such anomeric forms are also referred to as anomers [1,10]. Mutaro-Spatial isomers are also called stereoisomers, which differ from each other in the arrangement of their atoms in space [1]. The two types of stereoisom-erism are configurational and conformational isomstereoisom-erism. Configurational ste-reoisomers of monosaccharides are based on the existence of chiral carbon atoms in their molecular structure. Monosaccharides with three to nine carbon atoms contain one to seven chiral carbon atoms. A chiral, asymmetric carbon atom to which four different groups are attached can exist in two different configurations which are mirror-images of each other [10]. Such configura-tional stereoisomers, where analogous chiral carbon atoms have the opposite configuration, are referred to as diastereoisomers or epimers [1]. All of the monosaccharides are epimers of other monosaccharides; for example, D -glu-cose is a 2-epimer of D-mannose, a 3-epimer of D-allose, a 4-epimer of D -galactose, and a 5-epimer of L-glucose. For a hexose (such as glucose) con-taining 4 asymmetric carbon atoms, 16 (= 24) diastereoisomers are possible, 8 of which belong to a chiral family of D-sugars and 8 to a chiral family of
L-sugars. A sugar belongs to the D or L family of sugars when its highest numbered chiral carbon atom (carbon 5 in glucose) has the hydroxyl group tation is the interconversion of these α and β anomer forms (see Section 2.3.2).
written to the right or left, respectively, in the Fischer projection formula [1].
Monosaccharides can also be enantiomers, which are configurational stereoi-somers bearing a total mirror-image relation to each other; for example, the enantiomer of D-glucose is L-idose.
Conformational stereoisomerism involves the cyclic forms of monosac-charides [1,10]. For example, β-D-glucopyranose can occur in various confor-mations, or shapes — two “chair” conformations (with three carbon atoms and the ring oxygen in a plane and two carbon atoms positioned one above and the other below the plane) and various “boat” conformations (with four atoms of the ring in a plane and two atoms of the ring positioned either above or below the plane). Such conformations are energetically favored when bulky groups (hydroxyl and hydroxymethyl groups) are in the equatorial positions, such as the 4C1 chair conformation of β-D-glucopyranose.
Disaccharides are sugars with a molecular formula of Cn(H2O)n–1. They consist of two monosaccharide units condensed with the concomitant loss of one molecule of water [9]. Disaccharides may be homogeneous, having two similar monosaccharide units such as maltose (4-O-α-D-glucopyranosyl-D -glucopyranose) and α,α-trehalose (α-D-glucopyranosyl-α-D -glucopyrano-side), or heterogeneous, having two different monosaccharide units such as lactose (4-O-β-D-galactopyranosyl-D-glucopyranose) and sucrose (α-D -glu-copyranosyl-β-D-fructofuranoside) [1]. There are differences in the chemical and physical properties of various disaccharides, although the molecular weights are the same.
Monosaccharides and disaccharides may have a free hemiacetal group in their structure, and such mono- and disaccharides are referred to as reducing sugars [1,9]. In nonreducing disaccharides (e.g., sucrose and α,α-trehalose), both anomeric hydroxyl groups participate in the formation of glycosidic linkage between two monosaccharide units. Reducing sugars take part in nonenzymatic browning, which is known as the Maillard reaction or the carbonyl–amine reaction [1]. The relative reactivity of reducing sugars in nonenzymatic browning has been found to increase with decreasing molecular weight. Pentoses are more reactive than hexoses, which are more reactive than reducing disaccharides [1].
2.3 MONO- AND DISACCHARIDES IN WATER SOLUTIONS
2.3.1 SOLUBILITY
According to Hogan and Buckton [11], dissolution involves the disruption of bonding between the solid molecules and the formation of bonds between the solute and the solvent. All the mono- and disaccharides are soluble in water, and most of them have a relatively high degrees of solubility [1]. The solubility
Mono- and Disaccharides: Physicochemical and Functional Aspects 45
concentration of the solute can be defined as the concentration resulting from maximum solubilization of the solute at a given temperature. In solution at the solubility concentration of the solute, the equilibrium condition between the solid and liquid phases of solute prevails; that is, the chemical potentials of the solute molecules in both liquid and solid phases are equal [8]. The water solubilities of various sugars differ, however, and an increase in temperature results in increased solubilities of various sugars [1,8,12]. The presence of various sugars in solution decreases the solubility of a sugar [1]; for example, in solutions containing both lactose and sucrose, the solubility of lactose was found to decrease with an increasing content of sucrose [13].
Gao and Rytting [14] reported that the heat of solution of crystalline sucrose was 17.3 J g–1, whereas that of amorphous, freeze-dried sucrose was –43.4 J g–1, determined using solution calorimetry at 25°C. Hogan and Buckton [11] reported corresponding values for crystalline and amorphous lactose; the heat of solution of α-lactose monohydrate was 56.2 J g–1 whereas that of amorphous, spray-dried lactose was –56.5 J g–1. They found that spray-dried lactose dissolved more rapidly in water than α-lactose monohydrate when the dissolution of the mixture containing 50% α-lactose monohydrate and 50%
spray-dried lactose was studied at 25°C.
Gao and Rytting [14] and Hogan and Buckton [11] found that the enthalpy of solution increased linearly with a decreasing content of amorphous sugar and an increasing content of crystalline sugar in the mixture. Hogan and Buckton [11] suggested that solution calorimetry can be used in the quantifi-cation of relatively small contents (from 1 to 10%) of amorphous material in predominantly crystalline material assuming that the enthalpies of solution of the amorphous and crystalline forms differ.
2.3.2 MUTAROTATION
Mutarotation of a reducing sugar in solution may involve five structural iso-mers: α- and β-pyranose, α- and β-furanose, and the aldehydo or keto (open-chain) form [1]; however, the presence of all the various isomers at the same time is uncommon. For example, mutarotation of D-glucose at 20°C results in a mixture containing 36% α-D-glucopyranose and 64% β-D-glucopyranose because of the instability of the furanose forms and the very low concentration of the open-chain form [1,12]. Mutarotation of a reducing sugar in solution can be observed using a polarimeter because mutarotation causes changes in the optical rotatory power due to changes in the amounts of the anomeric forms of sugar [1]. Mutarotation occurs until an equilibrium ratio of anomeric forms is achieved. The rate of mutarotation increases with increasing temper-ature [1]. According to Hartel [8], the rate of mutarotation is a complex function of solution conditions, such as temperature, concentration, pH, and the presence of impurities.
Specific optical rotations have been reported for anhydrous α-lactose and β-lactose, as well as lactose, in equilibrium solutions at various temperatures [15,16]. The equilibrium ratio of β and α anomers of lactose was found to
Specific optical rotations have been reported for anhydrous α-lactose and β-lactose, as well as lactose, in equilibrium solutions at various temperatures [15,16]. The equilibrium ratio of β and α anomers of lactose was found to