O THER M ICROBIAL P OLYSACCHARIDES

5.2.3 O THER M ICROBIAL P OLYSACCHARIDES

A significant group of biopolymers with biological activities are glycans. Glycans are polysaccharides with molecules other than glucose in their main chain. 5 They

are classified as mannans, galactans, xylans, fructans, and fucans, according to the sugar units of the chain backbone. Heteroglycans contain combinations of the above

sugars and side chains of mannose, galactose, fucose, xylose, arabinose, glucose, and glucuronic acid. 5

Levans are representative immunomodulatory glycans. They are extracellular polysaccharides composed solely of fructose (fructans). They are produced by the bacteria Zymomonas mobilis 85 and Aerobacter levanicum. 86 Catalazans et al. purified

Microbial Polysaccharides

several levan fractions, with weight-average MW ranging from 3.5 to 10.7 ×10 5 Da. 87 The same authors suggested that a levan fraction with an MW of ~5 × 10 5 Da had the highest antitumor activity. Since Z. mobilis is also a large ethanol producer in the industry, levans could be utilized as valuable by-products of ethanol distilleries that are already in place. Alternatively, new processes designed for maximal levan productivity from Z. mobilis should aim at minimizing ethanol biosynthesis. 88

Several fungi also produce glycans with immune-enhancing effects. A bioactive galactomannan has been isolated from fruit body extracts of a common, edible mushroom, Morchella esculenta. 89 It is composed of major residues of mannose (62.9% molar content) and galactose (20.0%), and minor residues of N-acetyl glu- cosamine (7.9%), glucose (6.5%), and rhamnose (2.7%). The MW of the biopolymer

was estimated to be ∼10 6 Da. 89 This high MW glycan was clearly different from three heteropolysaccharides obtained from liquid fermentation media of M. escu- lenta ; the latter isolates were of low MW (11.5 to 44 × 10 3 Da) and have not been reported to exert any immunostimulating effects. 89 Fruit bodies of the mushroom Sarcodon aspratus also contain a highly branched immunomodulating fucogalacatan with unusual structure. It is made up of a main chain of α-(1→6)-linked galactopyranose with side groups of β-(1→2)-galactopyr- anosyl, as well as side residues of α-(1→2)-L-fucosyl-α-(1→4)-D-galactopyra-

nose. 90 The postulated structure of this biomolecule is shown in Figure 5.4. 90 Other therapeutic-immunopotentiating mushroom glycans include an arabinogalactan from Pleurotus citrinopileatus , 91 a mannofucogalactan from Fomitella fraxinea, 92 a man- nan, 93 a fucomannogalactan from Dictyophora indusiata, 94 a mannogalactan from Pleurotus pulmonarius , 95 a mannogalactofucan from Grifola fondosa, 96 and a xylan from Hericium erinaceus. 97 Alginates are a family of microbial polysaccharides with multiple dietetic (reg- ulation of lipid and glucose metabolism) and curative properties. They are produced either by brown (mainly) and red algae or by the bacteria Azotobacter vinelandii and Pseudomonas aeruginosa. 98,99 In the present chapter, the focus will be on the bacterial alginate, and in particular that from A. vinelandii, since P. aeruginosa is a pathogenic microorganism and its use in food applications is unlikely. However, much of the information registered herein on the chemical and physiological prop- erties of bacterial alginate is also valid for alginates from seaweed sources. Alginic acid and the sodium, calcium, potassium, and other salt forms are safe for use in

4 ϭ6)-α-Galp-(1ϭ

1 β-Gal

FIGURE 5.4 Postulated chemical structure of fucogalactan from Sarcodon aspratus. (From Mizuno, M. et al., Immunopharmacology, 46, 113, 2000. With permission.)

Functional Food Carbohydrates

food (GRAS) and are used in foodstuffs as thickeners, stabilizers, or gelling agents. Typical examples of use of alginate in food are in the production of jams, sweets, juices and soft drinks, ice cream, soups, sauces, margarine, milk shakes, liquors,

structured meat, milk, and fish products. 99 Recent research has revealed a number of biological effects of alginates, which, in combination with the multiple existing

food applications of alginate, render these biopolymers potential key ingredients in the manufacture of functional foods or neutraceuticals.

In terms of chemical composition and structure, alginates are unbranched, binary co-polymers of (1 →4)-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) with varying composition (content of the M and G groups) and chain length. 98,99 Also, microbial alginates are acetylated on some mannuronic acid residues. 99 Homopolymeric M and G regions are normally interspaced with alternating residues of both acids (MG groups) (Figure 5.5). Both the sequence of the two acids in the MG region and the length and frequency of the G block are characteristic for

Azotobacter alginate. 99 Alginate biosynthesis is initiated with the accumulation of poly-D-manuronic acid polymer extracellularly, which is then partly converted into poly-L-guluronic acid through the action of an extracellular C-5-epimerase. The activity of this enzyme depends on the presence of calcium ions. Calcium is also vital for the formation of the characteristic structure of alginate gels. The metal ions bind to the carboxyl and hydroxyl groups of adjacent guluronic acid residues, resulting in the so-called egg-box conformation (Figure 5.5). 98–101 Alginate gels

FIGURE 5.5 Structure of Azotobacter vinelandii alginate. (a) Block structure. (b, c) The calcium ion-dependent epimerization process and the formation of gel, according to the so- called egg-box model. (From Sabra, W. et al., Appl. Microbiol. Biotechnol., 56, 315, 2001. With permission.)

Microbial Polysaccharides

cannot be formed unless the guluronic acid content of alginate is at least 20%, indicating the important role of guluronic acid in the physicochemical properties of the biopolymer. 98

Last but not least, xanthan, a well-known gum in the food industry, is another microbial polysaccharide with reported biological functions. Xanthan is produced by the phytopathogenic bacterium Xanthomonas campestris and consists of glucose, mannose, and glucuronic acid as major components, and pyruvate and acetate as minor components. 102 Structurally, it is characterized by a cellulosic (D-glucosyl) backbone, with a trisaccharide side chain of internal D-mannose, D-glucuronic acid, and external (terminal) D-mannose linked on every second glucose molecule of -(1 →4) cellulosic backbone. Pyruvic acid diketal groups (pyruvate) are fixed to the 4,6-position of the terminal mannose residues of the trisaccharide side chain, while O-acetyl groups are located at the 6-position of the internal mannose. 102 Although there is some variance in the reports on the molecular weight of xanthan,

this is usually in the range of 4 to 12 × 10 6 Da. 103 Xanthan possesses Food and Drug Administration (FDA) approval for use in food and currently dominates the food market in the field of thickeners, stabilizers, and gelling agents, due to the high yields and low production cost of the biopolymer. 104 Despite the potential functional properties of xanthan, as displayed by some researchers in experiments with rats, 105 Castro et al. questioned any practical physiological effects of xanthan in humans, arguing that high doses of xanthan, incompatible with the food appli- cations, are probably needed to bring about the desired biological effects. 106 Thus, in the absence of clinical experiments on humans, it is still ambiguous whether or not xanthan can be established as a functional ingredient. More research on this matter is needed, since the possible biological effects of such a widely used food additive would have a great impact on the dietetic value of many foodstuffs.