**1. Introduction**

46 The Complex World of Polysaccharides

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Energy is stored in plants in form of carbohydrates, including sucrose (i.e., saccharose, a disaccharide) and starch (a polysaccharide). Sucrose and starch are derived from glucose, the product of carbon dioxide and water in photosynthesis. These carbohydrates are not only essential as energy supplies in food and animal feed, but in recent years have also been tapped as a renewable fuel energy source by the fuel ethanol industry. Some plants, like sugar cane or sugar beet, produce juice rich in sucrose, a mixed glucose-fructose disaccharide. Brewer's yeast (*Saccharomyces cerevisiae*) can directly ferment sucrose into ethanol, whereas starch, like that found in corn or potatoes, must first be hydrolyzed, to glucose or fermentable glucose oligosaccharides by an enzyme (e.g., amylase) before fermenting into ethanol. Cellulose, structural polysaccharides in plants, is even more difficult to hydrolyze to glucose. To obtain fermentable sugars, cellulose-containing plant material is usually subjected to a physicochemical pretreatment followed by enzymatic hydrolysis with cellulases. Such pretreatment processes are expensive and not yet used commercially on a large scale.

Because sugar and starch are major components of human food, and cellulose is the principal component of ruminant feed, their large-scale use in fuel alcohol production is already affecting food prices in the world. Brazil began producing fuel ethanol from sugar cane 36 years ago and in 2010 supplied enough ethanol to sell gasoline containing 25% ethanol throughout the country. Similarly, a large-scale, government sponsored program of fuel ethanol production from starch, using maize as its source, was started in the United States in 2007. This industry has created vast quantities of yeast byproducts; including a commercial-scale source of yeast extract, yeast glucose and mannose polysaccharides. Sugar cane juice and sugar beet juice fermentations are especially convenient for yeast collection,

© 2012 Kwiatkowski and Kwiatkowski, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

because the post-fermentation solution does not contain solids such as non-fermentable residues, like those produced during corn mash fermentation.

The yeast cell wall contains three major constituents: glucan (glucose polysaccharide), mannan (mannose polysaccharide) and a protein fraction. The separation of these natural polymers is simple and inexpensive. However, it is difficult and expensive to obtain more than 65% pure fractions and, therefore, these components are produced and sold at these low levels of purity. Their largest commercial application has been as animal feed nutritional supplements (see, http://www.Alltech.com).

There are three well known types of glucose polysaccharides in yeast: poly-(1→3)(1→6)-β-D-glucopyranose - commercial name: yeast β-D-glucan, yeast glucan; poly-(1→4)-α-Dglucopyranose (commercial name yeast glucogen) and poly-(1→6)(1→3)(1→4)-α-Dglucopyranose a recently "rediscovered" polysaccharide (Arvindecar & Patil, 2002) yet without a commercial name. The prefix "poly" informs about the polymeric nature of the material built of D-glucose cyclic monomers (D-glucopyranose rings) and the letter D informs that glucose belongs to the group of naturally occurring plant sugars with D stereochemistry. The letter α or β refers to the configuration of the glycosidic bonds between the C-1 of a nonreducing ring and the C-6 of a reducing ring or the C-1 of a nonreducing ring and the C-3 of a reducing ring (see Fig. 1, Lindhorst 2003). Figure 1 shows structures of β-D-glucopyranose isomer with a C-1 OH group in the β position (solid line) or a C-1 OH group in the (α) position (dotted line). Penta-(1→6)(1→3)-β-D-glucopyranose drawing shows the location of all OH groups, glycosidic bonds and glucopyranose rings within the structure. (The hydrogen atoms attached to carbon atoms are not shown.)

#### **Figure 1.**

Solving polysaccharide structure is not simple. At first, a sample needs to be homogenous and 98-99% pure. Different separation techniques (i.e., size exclusion chromatography, affinity (lectin) chromatography and in some cases high performance liquid chromatography (HPLC) are suitable for the purification of water or solvent soluble samples. Spectroscopic methods, such as 1H and 13C nuclear magnetic resonance (NMR) 1D/2D and mass spectrometry (MS) combined with chemical treatments of the sample, can provide further clues about the kind, sequence and ratios of the monosaccharides present, the type of bonds (α- or β-) between individual sugars, the branching points and reducing and non-reducing ends, and its molecular weight.

The experimental procedures that are most frequently used in the process of solving the polysaccharide structure are:

1. Sample purification

48 The Complex World of Polysaccharides

non-reducing end

O

O

HO OHHO

HO

HO HO

OH

Fig. 1

**Figure 1.**

HO HO

because the post-fermentation solution does not contain solids such as non-fermentable

The yeast cell wall contains three major constituents: glucan (glucose polysaccharide), mannan (mannose polysaccharide) and a protein fraction. The separation of these natural polymers is simple and inexpensive. However, it is difficult and expensive to obtain more than 65% pure fractions and, therefore, these components are produced and sold at these low levels of purity. Their largest commercial application has been as animal feed

There are three well known types of glucose polysaccharides in yeast: poly-(1→3)(1→6)-β-D-glucopyranose - commercial name: yeast β-D-glucan, yeast glucan; poly-(1→4)-α-Dglucopyranose (commercial name yeast glucogen) and poly-(1→6)(1→3)(1→4)-α-Dglucopyranose a recently "rediscovered" polysaccharide (Arvindecar & Patil, 2002) yet without a commercial name. The prefix "poly" informs about the polymeric nature of the material built of D-glucose cyclic monomers (D-glucopyranose rings) and the letter D informs that glucose belongs to the group of naturally occurring plant sugars with D stereochemistry. The letter α or β refers to the configuration of the glycosidic bonds between the C-1 of a nonreducing ring and the C-6 of a reducing ring or the C-1 of a nonreducing ring and the C-3 of a reducing ring (see Fig. 1, Lindhorst 2003). Figure 1 shows structures of β-D-glucopyranose isomer with a C-1 OH group in the β position (solid line) or a C-1 OH group in the (α) position (dotted line). Penta-(1→6)(1→3)-β-D-glucopyranose drawing shows the location of all OH groups, glycosidic bonds and glucopyranose rings within the

OH

reducing end

OH


HO O

OH

O

OH H

<sup>1</sup> <sup>6</sup>

O

HO

3

HO HO

OH

1

<sup>O</sup> <sup>O</sup>

OH

Solving polysaccharide structure is not simple. At first, a sample needs to be homogenous and 98-99% pure. Different separation techniques (i.e., size exclusion chromatography, affinity (lectin) chromatography and in some cases high performance liquid chromatography (HPLC) are suitable for the purification of water or solvent soluble

<sup>O</sup> HO

<sup>1</sup>

OH

structure. (The hydrogen atoms attached to carbon atoms are not shown.)

non-reducing end

OH

O O

residues, like those produced during corn mash fermentation.

nutritional supplements (see, http://www.Alltech.com).

A solid, crude, non-homogenous, polysaccharide must be solubilized in an appropriate solvent (water) or, in the case of insoluble polysaccharides, it must be subjected to chemical or enzymatic degradation (with minimal damage to the original structure) to reduce the polymer size which then allows the sample to become soluble. Sample solubilization makes the use of various chromatographic techniques (i.e., ion exchange chromatography, size exclusion chromatography, affinity-chromatography, sorbent-liquid chromatography) for sample fractionation and purification, possible.

2. Composition analysis

Complete acid hydrolysis is used to cleave all glycosidic bonds in the polysaccharide and to release D-glucose as a single product (in the case of glucans), which is then identified by comparison to a D-glucose standard.

3. Methylation linkage analysis

All of the polysaccharide hydroxyl (OH) groups are methylated using an excess of methylation reagent (dimsyl sodium followed by methyl iodide). Obtained this way, the fully O-methylated derivative is then subjected to acidic hydrolysis of all glycosidic linkages. The released O-methylated derivatives of D-glucose: 2, 3, 4, 6-O-tetramethyl-Dglucose; 2, 3, 4-O-trimethyl-D-glucose; 2, 4-O-dimethyl-D-glucose are identified and quantified using HPLC/MS and appropriate standards. In the case of pentasaccharide (Fig.1) the ratios of the methylation products would be 2:2:1.

4. Spectroscopy

Finally, 1H and 13C NMR and M S are used to establish the sequence of glucopyranose rings, the position of branching points, the number of non-reducing ends and the nature of glycosidic bonds (α or β). If the polysaccharide contains repeating sequences, their ratios can be also established.

5. Enzymatic action

Glycosidases and lectins of known specificity are applied to confirm glucose oligosaccharide structures, from which the complete polysaccharide architecture can be deduced.

The Complex Carbohydrates Research Center located at The University of Georgia (http://www.ccrc.uga.edu) offers services covering all of the aspects of the polysaccharide structural assignments.
