STRUCTURE–ACTIVITY RELATIONSHIPS

5.5 STRUCTURE–ACTIVITY RELATIONSHIPS

The determination of the relationship between chemical structure and physical properties and the functionality of bioactive microbial polysaccharides is of great importance for understanding the mechanism of action and the targeted improve- ment of these biopolymers (by regulation of bioprocess conditions, chemical modification–derivatization, or genetic engineering methods), as well as for the preservation of the beneficial activities in a food environment and through food processing. Nevertheless, great controversy exists over this relationship, and vari- able results concerning structure–activity links have been published for several polysaccharide isolates.

The most important attributes that influence the activity of microbial polysac- charides are the molecular weight, degree and type of branching, existence of

conformational order (random coil vs. single, double, or triple helix), chemical substitution of polysaccharide derivatives, and solubility.

Many researchers suggest that the β-(1,3) linkage of the main chain is vital for the expression of antitumor activity by glucans. Glucans containing mainly (1,6) linkages generally exhibited lower antitumor activity than glucans with a (1,3)-linked

backbone. 5 This might be explained to some extent by the presence of a β-(1,3)- glucan receptor in macrophages, which enables macrophage activation by β-(1,3)- glucans. However, immunoactive glucans with a β-(1,6)-linked backbone have also

Microbial Polysaccharides

been isolated, which shows that the β-(1,3)-linked main chain is not always a prerequisitive for antitumor activity of glucans. 2,5,57

The degree of branching (DB) also affects the biological functionality of polysaccharides. Table 5.1 demonstrates that glucans with low, medium, or high DB may possess immunostimulatory properties. For antitumor activity of glucans,

perhaps a moderate branching (DB between 0.20 and 0.33) is preferable. 2 The highly branched glucans of Auricularia auricula-judae (DB = 0.75) and Pestalotia sp. 815 (DB = 0.67), as well as the almost linear glucan of Ganoderma lucidum (DB = 0.06), are weakly active against tumors. 2,19,29,49 However, native PGG had low immunopotentiating activity when DB was 0.2, but genetically engineered PGG with a DB of 0.5 was more effective and showed a 35-fold higher affinity

for the β-glucan receptor of human monocytes and neutrophiles. 28 Increased branching leads to weaker interchain associations, which favors the formation of single helices. These structural differences resulting from different DBs may relate to the variance in biological activity of polysaccharides of different origin and

with different DBs. 28 Cleary et al. 203 found that between two modified β-D-glucans prepared from S. cerevisiae, those with more β-(1,6) side chains (20% (w/w) of the total molecule) led to higher stimulation of macrophages than their less branched counterparts (5% branches). This was ascribed to the higher ability of (highly) branched glucans to cross-link glucan receptors in macrophages, thus increasing the immunostimulatory effect. 203

The absence of branching often impedes immunomodulatory activity (e.g., of curdlan), because long, unbranched polysaccharides tend to be insoluble, and solu- bility seems to play an important role in polysaccharide–cell interactions. Interest-

ingly, branched (modified) curdlans exhibited significant antitumor activity. 2 Never- theless, branched curdlans did not have the ability to stimulate macrophages and

induce phagocytosis, 181 which indicates that the structural requirements (of polysac- charides) for stimulation of immune cells may be different than those for antitumor activity or other bioactivities.

The effect of molecular weight on the biological functionality of microbial polysaccharides is also surrounded by some controversy. High MW usually promotes antitumor and immunomodulating activity of glucans. It has been suggested that size (MW) is more important than structure in microbial glucans, because they

usually act via a nonspecific immune mechanism. 2 Furthermore, molecular weight can affect the ordered conformation of glucans: high MW schizophyllan (above

100,000 Da) exists in a triple helical structure, whereas low MW schizophyllan (below 50,000 Da) forms a single helix, but not a triple helix. 204 Schizophyllan 204

and the glucans isolated from Phytophthora parasitica 30 exhibit high antitumor activity when their MW is high (100,000 to 200,000 Da), while their lower MW fractions (5000 to 50,000 Da) are reported to be biologically inactive. On the contrary, the low MW (10,000 to 20,000 Da) β-glucans of Pythium aphanidermat-

icum have significant antitumor effects. 11 A positive link between high MW and immunomodulatory activity of S. cerevisiae particulate β-D-glucans was observed when glucans with an MW of 500,000 to 4,000,000 Da were compared. 203 Con- versely, Sandula et al. 169 reported that solubilized (carboxymethylated and sulfoet- hylated) glucans of the same yeast had high mitogenic, radioprotective, and antimu-

Functional Food Carbohydrates

tagenic activity when their MW was reduced ultrasonically (to 90,000 to 100,000 Da, from an initial 300,000 to 600,000 Da). The contradiction in the last two examples suggests that the way MW affects the biological functionality of glucans depends partly on the solubility of the molecule.

In variance with β-(1,3)-glucans, the immunopharmacological functionality of heteroglycans is usually not (strongly) influenced by differences in MW. 43,67,205 The

ability of α-(1,3)-glucuronoxylomannans of Tremella fuciformis to induce interleu- kin-6 production by human monocytes resulted mainly from the existence of an α- (1,3) backbone and was barely influenced by the molecular weight of the biopoly- mer. 205 Tumor inhibition by levans (fructans) of Zymomonas mobilis was dependent

on MW, with the molecular fraction of 211,000 Da being the most active. 87 Levan fractions with higher or lower MW were less active, which suggests that the isolation and purification procedures have an impact on obtaining the most preferable molec- ular fractions.

The bioactivity of alginates is also affected by the molecular size of the polysac- charide. High MW is desirable for traditional food applications where it is used as

a thickener or gelling agent, but low MW alginates are more soluble, and thus preferable when solutions of low viscosity are prepared. 206 Kimura et al. 206 studied the functional properties of sodium alginates of 10,000 (AG-1), 50,000 (AG-5), and 100,000 (AG-10) Da produced from a natural, high MW sodium alginate (2,700,000 Da, AG-270) after heat treatment at 130°C (for 250, 120, and 100 min, respectively). They found that AG-5 and AG-10 increased cholesterol excretion into feces and lowered blood glucose and insulin levels in a manner similar to that of natural alginate (AG-270), while AG-5 intake reduced blood glucose but did not affect insulin levels. AG-1 increased neither cholesterol excretion nor glucose tolerance. This implies that intense and prolonged heat treatment of alginate in a food envi- ronment should be avoided for its functional properties to be preserved.

The composition of alginate is of even greater importance for its bioactivity and functionality. High content of mannuronic acid is connected to higher induction of cytokinesis and antitumor activity, while guluronic acid appears to be nonstimulating for the immune system. 98,99 In fact, it has been claimed that in pharmaceutical preparations (pure solutions), guluronate should be eliminated because it may cause unwanted side effects, such as antibody generation. 101 On the other hand, guluronate is much more effective in binding radioactive molecules and heavy metals than mannuronate, and alginate with a high (G)/(M) ratio is an effective antitoxic, radio- protective agent. For example, a single administration of sodium alginate enriched with guluronic acid caused a fourfold decrease in the retention of strondium in

humans. 98 Furthermore, an animal diet with alginate rich in guluronic acid resulted in reduced consumption of food and slower increase of body weight in comparison with a diet with mannuronate-rich alginate. However, liver adiposity could be better controlled with consumption of alginate saturated with mannuronic acid. 98

The structural conformation and the presence of an ordered or nonordered structural domain play a substantial role in the functionality of many bioactive microbial polysaccharides, although different biopolymers are affected in different ways. The ordered structure in medicinal mushroom β-(1,3)-glucans is often

considered essential for their immunostimulating functions. 5 When the triple helix

Microbial Polysaccharides

of lentinan was denatured with dimethyl sulfoxide (DMSO), urea, or NaOH, the tertiary structure was lost and the antitumor activity was lowered, according to the degree of denaturation. 207 The loss of triple helical structure was also detri- mental for the immunological effects of schizophyllan. 208 In a complete reversal of these results, Saito et al. 209 concluded that lentinan and schizophyllan were better immunostimulants when they acquired a single-helix conformation. These differences imply that a single helix and triple helix may both have immunostim- ulatory effects, but they have different modes of action and affect the immune system in different ways. For instance, macrophage nitric oxide synthesis and limulus factor G activation are dependent on this tertiary structure. However, other immunological effects, such as interferon synthesis and colony stimulating factor, do not depend on the tertiary structure. 5

In other studies, immunostimulation by antitumor polysaccharide from Pythium aphanidermatum was higher when the biopolymer had a single helical conforma- tion, 11 while the antitumor effects of glomerellan were independent of ordered structure. 210 Kulicke et al. examined scleroglucan from Sclerotium rolfsii and Scle- rotium glucanicum and the fungal glucans synthesized by Monilia fructigena and Monilia fructicola and supported that immunological activity did not require or was not favored by helically ordered structures (the coil-like scleroglucan from S. rolfsii was the best activator of human blood monocytes). 211 Suzuki et al. 212 attempted to elucidate the impact of ordered structure on the immunological characteristics of microbial glucans such as curdlan, grifolan, schizophyllan, and SSG-glucan. They found that the triple helix activated the Alternative Pathways of Complement (APC) in the immune system more strongly than did the single helix, while single helical conformation was more efficient in stimulating the Classical Pathways of Comple- ment (CPC). Activation of CPC by the single helix was dependent on the degree of branching and occurred via the binding of the glucan to immunoglobulin in serum. 212 Of course, both systems are crucial for the expression of immune responses, and structural conformation seems to influence the way glucans are recognized by each of these systems.

Another factor that influences the performance of bioactive microbial polysac- charides is their solubility and the chemical substitution (with sulfate, phosphate,

carboxymethyl, or hydroxyl groups) of the original biopolymer. The degree of branching (DB) or the degree of substitution (DS) enhances water solubility, but only to a certain limit. The antitumor activity of many glucans is promoted by high solubility, which seems to depend primarily on the number and type of hydrophilic (anionic) groups positioned on the outer surface of the helix. 2,169 Particulate β- glucans of S. cerevisiae were solubilized after ultrasonication and chemical deriva- tization (with carboxyethyl and sulfoethyl compounds). 169 Carboxymethylated glu- cans with DS ranging from 0.4 to 1.15 were water soluble, while samples with DS below 0.4 were insoluble or only partly soluble. Optimal biological activity of carboxymethylated glucans was in the DS range of 0.6 to 0.8, and the immunomod- ulating activity of carboxymethyl glucans with DS above 1.0 gradually diminished.

A comparatively lower DS was needed to achieve solubility of sulfoethyl S. cerevi- siae glucans; glucans with DS = 0.3 were totally soluble when sulfoethyl groups were introduced, indicating the importance of the ionic substituents. 169

Functional Food Carbohydrates

The role of chemical substitution with ionic groups in enhancing biological activity has been demonstrated in many studies. Curdlan sulfate 2,189 and sulfated schizophyllans 213 inhibited the growth of HIV virus, depending on their sulfur content. In fact, the latter was the most determinant in HIV treatment by SPG, compared to the molecular weight or the chemical nature of the polysaccharide component. 213 Other examples of enhanced immunostimulation after solubilization by chemical substitution include the carboxymethylated β-glucan from Pleurotus ostreatus (pleuran) 214 and the carboxylated linear α-(1,3)-glucans from the fruit bodies of Amanita muscaria and Agrocybe aegerita. 215 Carboxymethylated SSG displayed antitumor properties with DSs up to 0.14, above which its activity grad- ually decreased. It was postulated that this might be due to changes in the electric charge that modulates the electrostatic binding of the polysaccharide to a receptor or ligant. 216

Another form of derivatized biopolymers with increased solubility and immu- nological activity are polyols. Glucan polyols or glucan polyaldehydes of grifolan, pestalotan, and Auricularia sp. glucan, formed after oxidation and subsequent reduc- tion of the original polymeric structure, were characterized by enhanced antitumor potency, which was a function of the polyol content. 5,19,27

Despite the above literature on the positive link between solubility and polysac- charide functionality, some microbial polysaccharides are more effective or only active in insoluble (particulate) form. For instance, the particulate β-(1,3)-glucan from Candida albicans cell wall was more active in stimulating human leukocytes,

specifically TNF-α, interleukin-α, and hydrogen peroxide production, 45 than dime- thyl sulfoxide-soluble samples. Also, lentinan was effective against mouse sarcoma

(80 to 98% inhibition), but hardly soluble in water. 174 In addition to this, particulate (and modified) glucans of S. cerevisiae that were insoluble even for low MW

preparations proved to be efficient stimulators of macrophages. 203 The presence of ionic groups not only favors the solubility of microbial polysac-

charides, but also changes their charge and perhaps their ability to bind to other molecules or cells. In LAB functional polysaccharides it appears that the presence of phosphate groups determines the extent of biological activity. Mitogenic activity was lost after dephosphorylation of Lactobacillus bulgaricus phosphopolysaccha- ride, while a similar but neutral EPS by the same microorganism had no mitogenic activity. Although the exact role of phosphate groups in LAB EPS is not clear, it may be connected to direct stimulation of B-cell mitogenicity, as was shown for inorganic sulfate, or the ability to adhere strongly to intestinal epithelian cells, as was evident for Lactobacillus cremoris phosphopolysaccharide. 76,82,84,186 It has to be noted, however, that it is still not known whether and to what extent these phosphate groups retain their functionality after interactions with other polysaccharides, or with proteins, or through pH changes, as might occur in a real food environment.

Although there is a fair amount of literature on the structure–functionality relationship of many microbial polysaccharides, as shown above, most of these studies examine these molecules from a pharmacological perspective, where the pure bioactive compound is tested in vitro or administered in soluble or particulate preparations directly into the body of an animal or patient. To date there has been only limited research on how these biopolymers act in a real food environment

Microbial Polysaccharides

(incorporated as food ingredients) and how food processing practices (pasteurization, freezing, etc.) and the interaction with other food compounds may affect their structure, MW, physicochemical characteristics, and eventually their functional prop- erties. These are parameters that have to be taken into account in designing and marketing functional food products with certain bioactive function.

Hromadkova et al. 163 compared the effect of different drying methods, namely, solvent exchange (GE), lyophilization (GL), and spray drying (GS), on the biological activity of the particulate form of glucans from S. cerevisiae. They observed that immunomodulatory activity was twice as high with GS as with GL and GE. Also, GS dispersion had the lowest apparent viscosity, which perhaps denotes a relation- ship between biopolymer functionality and rheological behavior of aqueous disper- sions of the polysaccharide isolates. Thus, it was concluded that spray drying is the most suitable method for preparing particulate β-glucan suspensions.

The effect of mechanical shear on polysaccharide MW and hydration properties is another important aspect. Although reports on microbial polysaccharides are scarce, it has been demonstrated for hypocholesterolemic cereal β-glucans that mechanical shear can reduce their molecular mass without having an adverse effect on their ability to reduce plasma cholesterol. 217 Other processing parameters such as temperature (heating), pressure, or combined thermomechanical treatments (e.g., extrusion) may change the structure, porosity, solubility, and water retention prop- erties of microbial polysaccharides added in food, as has been shown for polysac- charides of plant origin. 218,219