**2. Fungal and yeast cell wall, D-glucose homo- and heteropolysaccharides**

Fungi constitute an independent evolutionary group of eukaryotic organisms equal in rank to that of plants or animals. They can excrete hydrolytic enzymes that break down biopolymers (polysaccharides and proteins) of plant or animal origin and use sugars and/or amino acids for their nutrition. Yeast is a single-cell fungus and the *Saccharomyces cerevisiae* species (baker's or brewer's yeast) has been utilized by people from early human civilization in food and beverage preparation. Mushrooms are fungal fruiting bodies; edible mushrooms are taste improving, "healthy" food ingredients that are very popular on European and Asian menus. Extracts and dried powders made by processing "medicinal mushrooms" are important ingredients of traditional Eastern medicinal remedies.

Fungal cell walls are made of polysaccharides. Chitin, a homopolymer of (1→4)-β-D-Nacetylglucosamine and various glucans (homopolymers of D-glucose, with α- or βglycosidyl linkages between C-1 and C-2, C-3, C-4 and C-6 of glucopyranose rings) are the most frequently found building blocks used by nature in construction of the cell wall that separates the fungus from the environment. In yeast, α- and β-glucans and α-mannan are the major polysaccharides (chitin is present at 1-2%) that are utilized in cell wall construction. Their ratio changes with yeast strain and growth stage and is dependent upon growth conditions including oxygen and nutrient availability and the temperature and pH of the medium (Stewart & Russell, 1998). Table 1 shows the average abundance of yeast cell wall components.


Mannan structure can be represented by the following formula: poly-(1→2) (1→3) (1→6)-α-D-mannopyranose. This polysaccharide is built out of a long backbone chain of (1→6)-α-D-mannopyranose rings with short mannopyranose chains (one to four rings long, connected through (1→2) and (1→3)-α-glucoside bonds (Ballou, 1980).

**Table 1.** The cell wall components of *Saccharomyces cerevisiae* (Klis et al. 2002; Kath & Kulicke, 1999; Lessage & Bussey, 2006; Kwiatkowski et al., 2009)

The yeast cell wall forms a border that defines the yeast cell's dimensions and separates its organelles from the negative influences of the environment. The individual constituents of the cell wall connect to each other by covalent bonds forming a single supra-molecular biopolymer. To find the nature of these connections, as well as the individual polysaccharides structures, all of the architectural elements must be dissected, solubilized, purified and analyzed. In this process it is almost impossible not to damage particular structural elements and to afterwards distinguish between the original components of the cell wall and soluble polysaccharides that could be present in the cytoplasm or might be trapped (physically absorbed) within cell wall. More than 50 years of research were required to establish the structure of the major, high molecular weight yeast homo-polysaccharides (Bacon & Farmer, 1969; Aimaniada et al., 2009).

50 The Complex World of Polysaccharides

**polysaccharides** 

wall components.

**2. Fungal and yeast cell wall, D-glucose homo- and hetero-**

important ingredients of traditional Eastern medicinal remedies.

Fungi constitute an independent evolutionary group of eukaryotic organisms equal in rank to that of plants or animals. They can excrete hydrolytic enzymes that break down biopolymers (polysaccharides and proteins) of plant or animal origin and use sugars and/or amino acids for their nutrition. Yeast is a single-cell fungus and the *Saccharomyces cerevisiae* species (baker's or brewer's yeast) has been utilized by people from early human civilization in food and beverage preparation. Mushrooms are fungal fruiting bodies; edible mushrooms are taste improving, "healthy" food ingredients that are very popular on European and Asian menus. Extracts and dried powders made by processing "medicinal mushrooms" are

Fungal cell walls are made of polysaccharides. Chitin, a homopolymer of (1→4)-β-D-Nacetylglucosamine and various glucans (homopolymers of D-glucose, with α- or βglycosidyl linkages between C-1 and C-2, C-3, C-4 and C-6 of glucopyranose rings) are the most frequently found building blocks used by nature in construction of the cell wall that separates the fungus from the environment. In yeast, α- and β-glucans and α-mannan are the major polysaccharides (chitin is present at 1-2%) that are utilized in cell wall construction. Their ratio changes with yeast strain and growth stage and is dependent upon growth conditions including oxygen and nutrient availability and the temperature and pH of the medium (Stewart & Russell, 1998). Table 1 shows the average abundance of yeast cell

Component Cell wall mass

(1→3)-β-D-glucan 50-55 (1→6)-β-D-glucan 5-10 (1→4)-α-(1→3)-β-D-glucan 3-7\* Mannoprotein complex 35-40 Chitin 2

Mannan structure can be represented by the following formula: poly-(1→2) (1→3) (1→6)-α-D-mannopyranose. This polysaccharide is built out of a long backbone chain of (1→6)-α-D-mannopyranose rings with short mannopyranose

**Table 1.** The cell wall components of *Saccharomyces cerevisiae* (Klis et al. 2002; Kath & Kulicke, 1999;

The yeast cell wall forms a border that defines the yeast cell's dimensions and separates its organelles from the negative influences of the environment. The individual constituents of the cell wall connect to each other by covalent bonds forming a single supra-molecular biopolymer. To find the nature of these connections, as well as the individual polysaccharides structures, all of the architectural elements must be dissected, solubilized, purified and analyzed. In this process it is almost impossible not to damage particular structural elements and to afterwards distinguish between the original components of the

chains (one to four rings long, connected through (1→2) and (1→3)-α-glucoside bonds (Ballou, 1980).

Lessage & Bussey, 2006; Kwiatkowski et al., 2009)

(%, dry weight)

The (1→3)-β-D-glucan and (1→6)-β-D-glucan form a single structure in which the (1→3)-β-D-glucan forms the backbone chain with (1→6)-β-D-glucan branches that are attached to branching glucopyranose rings at C-6 (Lessage & Bussey, 2006). The (1→3)-β-D-glucan chains form triple helix tridimensional structures with spring-like mechanical properties, responsible for the yeast cell wall's strength (Klis et al., 2002) and its ability to absorb toxins (Yiannikouris et al., 2004). The (1→6)-β-D-glucan is a linker between (1→3)-β-D-glucan, chitin and mannoproteins (Kaptein et al., 1999; Kollar et al., 1997) that stabilizes the whole structure and is the main cause of yeast cell wall insolubility. The properties and role of a mixed (1→4)-α-(1→3)-β-D-glucan from yeast cells will be discussed in section 3 of this chapter.

Mannoproteins are mostly located on the outside surface of the cell wall (Osumi, 1998). They play a sensory function for nutrients and chemical and bacterial/viral toxins, actively participate in the transport of nutrients and metabolites through the cell wall, and also participate in mating (Klis et al., 2002; Stewart & Russell, 1998). Some cell wall enzymes such as glucanase, mannanase, invertase, alkaline phosphatase and lipase are mannoproteins (Stewart & Russell, 1998) that hydrolyze nutrients and participate in the reconstruction of cell polysaccharides during cell growth and budding. Cell wall mannans are connected with cell wall glucans via covalent bonds, but they can be released under action of an alkaline medium in which they are perfectly soluble. The process of separating yeast cell wall glucans (which stay as an insoluble fraction) from yeast mannans is used on an industrial scale (Sedmak, 2006). The soluble, mannan-rich fraction, can be added back to the cell wall which contains only 10-17% mannan by weight, to increase the content of mannan to 30% by weight. Such a product is sold by Alltech Inc. under the name of ActigenTM to the animal nutrition industry.

The small fraction of chitin present in yeast cell wall is primarily located in the scar rings around buds in budding yeast and secures closure of the gaps in the mother cell and the departing daughter cell (Lessage & Bussey, 2006; Stewart & Russell, 1998).

## **3. α-D-glucans from baker's yeast (***Saccharomyces cerevisiae***)**

The role of (1→3)-β-D-glucan in the maintenance of yeast cell wall shape and rigidity (Lessage & Bussey, 2006; Klis, et al. 2006) and (1→6)-β-D–glucan as a polysaccharide that links together all of the cell wall polysaccharides (Aimaniada et al., 2009; Kollar et al., 1997) is well documented and has been reviewed by Lessage & Bussey (2006) and Klis et al. (2002). The presence of starch-like, "alkali-soluble glycogen, "an "energy storage polysaccharide" in cell cytosol, and "difficult to dissolve acid-soluble," glycogen like (1→4)-α-D-glucan in the cell walls of *Saccharomyces cerevisiae* grown aerobically, was frequently mentioned in early yeast literature (Grba et al., 1976). The two forms of glycogen synthetase have also been identified (Rothman-Denes & Cabib, 1970; Lille & Pringle, 1980) but the yeast literature is still treating the "difficult to dissolve" α-glucan as physically adsorbed cytosol glycogen,

trapped within yeast cell wall, and not as an independent component attached by a covalent bond to the other cell wall polysaccharides. In 1973, Manners et al. reported: "β-1, 6-glucan purified after acid extraction had to be exhaustively treated with α-amylase and still showed the presence of glucose and maltose in paper chromatographic analysis, along with gentiobiose and higher gentiooligosaccharides," which are (1→4)-α-D-glucan oligosaccharides formed during enzymatic hydrolysis of the mixed α, β-glucan from the yeast cell wall.

The α-glucan content in the yeast cell wall is reported to vary from as little as 1% (Lille & Pringle, 1980) to as much as 29% (Sedmak, 2006) of the dry weight, depending upon the nutritional status of the cells, the method of isolation, the method of analysis and the phase of growth during which the cells were harvested (Lille & Pringle, 1980). Spectrophotometric analysis of soluble glycogen can be run directly on a water extract, and if properly done, yields reliable results (Quain, 1981). Quantitative analysis of "insoluble" glycogen requires additional, enzymatic release of glucose from its mixed polysaccharide with β-glucan (see, http://www.megazyme.com). The industrially produced brewer's yeast described in Sedmak's US patent application (Sedmak, 2006) contains glucans (α + β) at 28.9% dry weight, which includes 12.4% α-glucan. Hydrolysis of these water-insoluble cells with alkaline protease solubilizes the mannoprotein complex and yields water-insoluble cell wall polysaccharides (including α-glucan) that contain 54.5% dry weight of glucans with more than half of the total weight (29.2%) as α-glucan.

Arvindekar and Patil (2002) proposed an explanation for the presence of α-glucan in the insoluble fraction from yeast cell walls, which has been described by others as "difficult to wash away" yeast glycogen (Gunia-Smith et al., 1977; Manners & Fleet, 1976). They found that the ratio of "soluble" to "insoluble" glycogen in different strains of *Saccharomyces cerevisiae* is in the range of 1:2.5–1:3. When the insoluble fraction was digested with purified lyticase, which possessed only (1→3)-β-D–glucan hydrolyzing activity and no (1→6)-β-D– glucan or (1→4)-α-D-glucan hydrolyzing activities, all of the material dissolved. The affinity chromatography of the solution on a Concanavalin A (ConA) column retained a fraction that contained mixed (1→4)-α-(1→6)-β-D–glucan polysaccharide. The eluent contained exclusively (1→3)-β-D–glucan oligosaccharides. When the (1→4)-α-D-glucan-rich fraction was released from its binding with ConA and treated with amyloglucosidase followed by dialysis to remove glucose, subjecting the resulting solution to affinity chromatography on a ConA column showed none of the material was retained. The material recovered from the solution was then proved to be pure (1→6)-β-D–glucan. These simple experiments showed that the "insoluble glycogen" from yeast cell wall is a mixed glucan in which (1→4)-α-Dglucan is connected to (1→3)-β-D–glucan through a (1→6)-β-D–glucan link. The structure of this mixed yeast cell wall (1→4)-α-(1→6)-β-(1→3)-β-D-glucan was confirmed by our 1H NMR study (Kwiatkowski et al., 2009) and methylation analysis done at CCRC.

We used yeast cell wall α-glucan (prepared in our laboratory) in the development of the enzyme linked immunosorbent assay (ELISA) for yeast cell wall quantitative analysis in a complex polysaccharide matrix (Moran et al., 2011). The antigen was prepared in two stages. At first a sample of cell wall (1→4)-α-D-glucan (fraction 50-100 kDa separated with the help of an ultra centrifugal filter (Aldrich Inc.) was oxidized mostly at the C-6 carbons using the Albright-Goldman oxidation reagent (Albright & Goldman, 1967; Zekovic et al., 2006) which converted the –CH2OH groups into aldehyde groups –CH=O in glucopyranose rings. The reaction produced a highly cross-linked polymer, with internal acetal bonds, but still well soluble in DMSO (dimethyl sulfoxide) and water, which was coupled to a bovine serum albumin (BSA), yielding the antigen (glucan-BSA conjugate). To restore the original αglucan structure, but with –CH2–NH-BSA groups instead of –CH2-OH groups at the glucan's C-6 carbon, the conjugate C-6 imino (-CH=N-BSA) groups were reduced with sodium cyanoborohydride (Baxter & Reitz, 2002). Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) of the conjugate indicated the presence of significant quantities of free BSA (line at 66 kDa) together with the glucan-BSA conjugate of molecular size between 100 and 175 kDa. To remove the excess BSA, the concentrated solution of the conjugate was washed with water of pH 7.0, 8.5 and 4.5 using an ultra centrifugal filter with the 50-kDa membrane. This "purified" conjugate was used then in rabbit immunization. We found that the rabbit polyclonal antibodies were very sensitive to yeast cell wall α-glucan, but (as expected) they were also cross-reactive with BSA. To delete this activity we synthesized the BSA-coated affinity phase (Moran et al., 2011) and removed antibodies specific to BSA from the crude fraction of polyclonal rabbit anti-α-glucan antibodies. We also found that antibodies grown against this α-glucan polysaccharide did not recognize any commercially available starch or glycogen samples, including yeast glycogen. Such specificity confirms structural differences between the cell wall and the cytosol α-glucans from *Saccharomyces cerevisiae*. Because of the high specificity and very low cross reactivity of the "purified" rabbit antibodies with soy proteins and corn carbohydrates, the ELISA cell wall assay detects and quantifies samples of yeast cell wall down to 50 g/ton of feed (Moran et al., 2011). A similar assay was used for quantification of α-D-glucanprotein complex in mushrooms with immunomodulatory activity (Hirotaka et al., 2007).

52 The Complex World of Polysaccharides

than half of the total weight (29.2%) as α-glucan.

yeast cell wall.

trapped within yeast cell wall, and not as an independent component attached by a covalent bond to the other cell wall polysaccharides. In 1973, Manners et al. reported: "β-1, 6-glucan purified after acid extraction had to be exhaustively treated with α-amylase and still showed the presence of glucose and maltose in paper chromatographic analysis, along with gentiobiose and higher gentiooligosaccharides," which are (1→4)-α-D-glucan oligosaccharides formed during enzymatic hydrolysis of the mixed α, β-glucan from the

The α-glucan content in the yeast cell wall is reported to vary from as little as 1% (Lille & Pringle, 1980) to as much as 29% (Sedmak, 2006) of the dry weight, depending upon the nutritional status of the cells, the method of isolation, the method of analysis and the phase of growth during which the cells were harvested (Lille & Pringle, 1980). Spectrophotometric analysis of soluble glycogen can be run directly on a water extract, and if properly done, yields reliable results (Quain, 1981). Quantitative analysis of "insoluble" glycogen requires additional, enzymatic release of glucose from its mixed polysaccharide with β-glucan (see, http://www.megazyme.com). The industrially produced brewer's yeast described in Sedmak's US patent application (Sedmak, 2006) contains glucans (α + β) at 28.9% dry weight, which includes 12.4% α-glucan. Hydrolysis of these water-insoluble cells with alkaline protease solubilizes the mannoprotein complex and yields water-insoluble cell wall polysaccharides (including α-glucan) that contain 54.5% dry weight of glucans with more

Arvindekar and Patil (2002) proposed an explanation for the presence of α-glucan in the insoluble fraction from yeast cell walls, which has been described by others as "difficult to wash away" yeast glycogen (Gunia-Smith et al., 1977; Manners & Fleet, 1976). They found that the ratio of "soluble" to "insoluble" glycogen in different strains of *Saccharomyces cerevisiae* is in the range of 1:2.5–1:3. When the insoluble fraction was digested with purified lyticase, which possessed only (1→3)-β-D–glucan hydrolyzing activity and no (1→6)-β-D– glucan or (1→4)-α-D-glucan hydrolyzing activities, all of the material dissolved. The affinity chromatography of the solution on a Concanavalin A (ConA) column retained a fraction that contained mixed (1→4)-α-(1→6)-β-D–glucan polysaccharide. The eluent contained exclusively (1→3)-β-D–glucan oligosaccharides. When the (1→4)-α-D-glucan-rich fraction was released from its binding with ConA and treated with amyloglucosidase followed by dialysis to remove glucose, subjecting the resulting solution to affinity chromatography on a ConA column showed none of the material was retained. The material recovered from the solution was then proved to be pure (1→6)-β-D–glucan. These simple experiments showed that the "insoluble glycogen" from yeast cell wall is a mixed glucan in which (1→4)-α-Dglucan is connected to (1→3)-β-D–glucan through a (1→6)-β-D–glucan link. The structure of this mixed yeast cell wall (1→4)-α-(1→6)-β-(1→3)-β-D-glucan was confirmed by our 1H

NMR study (Kwiatkowski et al., 2009) and methylation analysis done at CCRC.

We used yeast cell wall α-glucan (prepared in our laboratory) in the development of the enzyme linked immunosorbent assay (ELISA) for yeast cell wall quantitative analysis in a complex polysaccharide matrix (Moran et al., 2011). The antigen was prepared in two stages. What is the role of this polysaccharide in yeast cell life? Some remarks in the literature point to its role in yeast flocculation (Patel & Ingledew, 1975), acting as part of a possible killer toxin receptor (Hutchkins & Bussey, 1983) and as being sensitive to environmental conditions and changes in medium (Slaughter & Nomura, 1992; Dake et al., 2010; Jadhav et al., 2008). The cell wall bound α-glucan from fission yeast *Schizosaccharomyces pombe* has been studied in detail (Villar-Tajadura et al., 2008; Vos et al., 2007 ; Garcia et al., 2006; Grün et al., 2005), and systematic identification of the genes affecting glycogen storage in the yeast *Saccharomyces cerevisiae* was attempted (Wilson et al., 2002). Unfortunately, the presence of the two different pools of glycogen was not addressed. One of the possible functions of bound α-glucan might be the temporary formation of mixed α, β-glucan oligosaccharides that become soluble in cytoplasm and can be transported inside the cell wall where the βglucan fragment is used in the construction of insoluble yeast cell wall β-glucan polysaccharide.

Is it possible that α-glucan polysaccharides from yeast or mushrooms can have medicinal properties? There is no literature concerning the medicinal properties of α-glucan as a

separate fraction of the yeast cell wall, which is a consequence of acknowledging it as part of an energy storage cellular polysaccharide-yeast glycogen. Bioactive components containing α-glucan, mixed α, β-glucan or α-glucan-protein complexes are known to be present in extracts from the fruiting bodies of edible medicinal-mushrooms. Like in the case of many other traditional herbal remedies, it is difficult to separate the single bioactive component's (α-glucan) bioactivity from the activities of the other components of the extract, and in some cases, early assignments were incorrect.

Mushrooms with medicinal activities that contain extractable α-glucan in their fruiting bodies, belong to one of two phyla: Ascomycota or Basidiomycota. Hot-water extracts from *Peziza vesiculosa* (Ohno et al., 1985; Suzuki et al., 1982) and *Cordyceps chinensis* (Yan et al., 2011; Holliday & Cleaver, 2008; Khan et al., 2010; Liu et al., 2006; Kiho et al., 1993; Kiho et al., 1996; Li et al., 2006), which are both Ascomycota, have been shown to have antitumor and anti-diabetic activities. The linear α-(1→4)-D-glucan is less active than α-(1→4) (1→6)-Dglucan with 1 to 6 branches on the backbone 1→4 chain with a ratio of 1:8. The biological activity of glycogen-like polysaccharides was found to be molecular-size dependent with smaller ~9.5 kDa molecules being active, whereas 14-24 kDa molecules were not (Kakutani et al., 2007).

More is known about edible mushrooms belonging to Basidiomycota which contain both: αand β-glucans in their fruiting bodies (Rop et al., 2009). Hot-water extracts from *Peziza vesiculosa* show antitumor activity (Ohno et al., 1985; Suzuki et al., 1982) and extracts from *Grifola frondosa* (Maitake) show glucose suppression (Tanaka et al., 2011) and antidiabetic (Lei et al., 2007) activities. Extracts (Shida & Matsuda, 1974) from *Lentinus edodes* (Shiitake) stimulate the immune system (Shah et al., 2011; Terakawa et al., 2008) and possess antibacterial, antiviral (Mach et al., 2008) and anticancer properties (Shida & Matsuda, 1974; Hyodo et al., 2005; Shah et al., 2011). Extracts from *Tricholoma matsutake* (Matsutake) show antitumor (Ebina et al., 2002) and immunomodulatory (Hirotaka et al., 2007; Hirotaka et al., 2005) activities. Extracts (Smiderle et al., 2010) from *Agaricus bisporus* (Portobello) inhibit breast cancer cell proliferation (Grube et al., 2001) and stimulate the immune system (Ren et al., 2008; Koppada et al., 2009). A glycogen-like polysaccharide from Portobello mushrooms potently activated macrophages, stimulating TNF-α production and phagocytosis of RAW264.7 cells (Kojima et al., 2010). Extracts from *Pleurotus ostreatus* (Oyster mushroom) show antiproliferative and proapoptotic activities on colon cancer cells (Lavi et al., 2006). *Pholiota nameko* (Butterscotch mushroom), which has been in use as a major component of miso soup in Japan, shows antinflammatory activity (Li et al., 2008). In Asian and European culinary traditions, meals containing these mushrooms are believed to be healthy and to heal a variety of ailments.

Whether α-glucan from yeast cell walls can contribute to the known medicinal activities of various extracts from whole yeast is still an open question. Newly developed, simple methods for producing large quantities of this material (Kwiatkowski et al., 2009; Moran et al., 2011) should stimulate new research in this area.

The abundant literature regarding bioactivity of the whole yeast cell wall and its extracts rich in α-mannoprotein or β-glucan, will be discussed in sections 4, 5 and 6 of this chapter.

#### **4. Baker's yeast cell wall β-D-glucan/α-mannoprotein complex**

54 The Complex World of Polysaccharides

et al., 2007).

heal a variety of ailments.

al., 2011) should stimulate new research in this area.

cases, early assignments were incorrect.

separate fraction of the yeast cell wall, which is a consequence of acknowledging it as part of an energy storage cellular polysaccharide-yeast glycogen. Bioactive components containing α-glucan, mixed α, β-glucan or α-glucan-protein complexes are known to be present in extracts from the fruiting bodies of edible medicinal-mushrooms. Like in the case of many other traditional herbal remedies, it is difficult to separate the single bioactive component's (α-glucan) bioactivity from the activities of the other components of the extract, and in some

Mushrooms with medicinal activities that contain extractable α-glucan in their fruiting bodies, belong to one of two phyla: Ascomycota or Basidiomycota. Hot-water extracts from *Peziza vesiculosa* (Ohno et al., 1985; Suzuki et al., 1982) and *Cordyceps chinensis* (Yan et al., 2011; Holliday & Cleaver, 2008; Khan et al., 2010; Liu et al., 2006; Kiho et al., 1993; Kiho et al., 1996; Li et al., 2006), which are both Ascomycota, have been shown to have antitumor and anti-diabetic activities. The linear α-(1→4)-D-glucan is less active than α-(1→4) (1→6)-Dglucan with 1 to 6 branches on the backbone 1→4 chain with a ratio of 1:8. The biological activity of glycogen-like polysaccharides was found to be molecular-size dependent with smaller ~9.5 kDa molecules being active, whereas 14-24 kDa molecules were not (Kakutani

More is known about edible mushrooms belonging to Basidiomycota which contain both: αand β-glucans in their fruiting bodies (Rop et al., 2009). Hot-water extracts from *Peziza vesiculosa* show antitumor activity (Ohno et al., 1985; Suzuki et al., 1982) and extracts from *Grifola frondosa* (Maitake) show glucose suppression (Tanaka et al., 2011) and antidiabetic (Lei et al., 2007) activities. Extracts (Shida & Matsuda, 1974) from *Lentinus edodes* (Shiitake) stimulate the immune system (Shah et al., 2011; Terakawa et al., 2008) and possess antibacterial, antiviral (Mach et al., 2008) and anticancer properties (Shida & Matsuda, 1974; Hyodo et al., 2005; Shah et al., 2011). Extracts from *Tricholoma matsutake* (Matsutake) show antitumor (Ebina et al., 2002) and immunomodulatory (Hirotaka et al., 2007; Hirotaka et al., 2005) activities. Extracts (Smiderle et al., 2010) from *Agaricus bisporus* (Portobello) inhibit breast cancer cell proliferation (Grube et al., 2001) and stimulate the immune system (Ren et al., 2008; Koppada et al., 2009). A glycogen-like polysaccharide from Portobello mushrooms potently activated macrophages, stimulating TNF-α production and phagocytosis of RAW264.7 cells (Kojima et al., 2010). Extracts from *Pleurotus ostreatus* (Oyster mushroom) show antiproliferative and proapoptotic activities on colon cancer cells (Lavi et al., 2006). *Pholiota nameko* (Butterscotch mushroom), which has been in use as a major component of miso soup in Japan, shows antinflammatory activity (Li et al., 2008). In Asian and European culinary traditions, meals containing these mushrooms are believed to be healthy and to

Whether α-glucan from yeast cell walls can contribute to the known medicinal activities of various extracts from whole yeast is still an open question. Newly developed, simple methods for producing large quantities of this material (Kwiatkowski et al., 2009; Moran et

The abundant literature regarding bioactivity of the whole yeast cell wall and its extracts rich in α-mannoprotein or β-glucan, will be discussed in sections 4, 5 and 6 of this chapter.

Yeast cell wall biogenesis was studied by Smits (Smits et al, 2001) who found that "The yeast cell wall forms a border that defines the yeast cell's dimensions and separates its organelles from the negative impact of the environment. All of the individual constituents of a yeast cell wall connect to each other by covalent bonds forming a single supra-molecular biopolymer". The (1→3) (1→6)-β-D-glucan and the (1→2) (1→3) (1→6)-α-D-mannan/protein complexes (Vinogradov et al., 1998) are the major components of this supra-molecular biopolymer (Lessage & Bussey, 2006; Kath & Kulicke, 1999). They connect to each other by a covalent bond the nature of which is still under investigation (Kaptein et al., 1999; Kollar et al., 1997). In the process of yeast cell wall isolation the cell wall is first cracked open, with the help of physical, chemical and/or enzymatic treatments (Kath & Kulicke, 1999) and then separated from the soluble yeast cell components by centrifugation (Sedmak, 2006; Jamas et al., 1998). The concentrated yeast cell wall solids containing ~30% dry weight material are then spray-dried. The resulting product is a fine powder with a light tan color. The cell wall polysaccharides can be separated from each other by alkaline extraction, which solubilizes the α-mannoprotein fraction and leaves β-glucan particles in suspension. The β-glucan particulate can be separated from the soluble α-mannoprotein fraction by centrifugation and spray-dried to yield light, yellow colored, fine powder, free of any smell or taste, containing ~65% β-glucan. The α-mannoprotein solution can then be concentrated by using membrane ultrafiltration and spray-dried to produce a light brown, "mannan rich fraction" with ~40% by weight of α-mannan. Yeast cell walls as well as both fractions of polysaccharides are produced on a large scale and have practical applications as animal feed nutritional supplements. Furthermore, this yeast cell wall preparation, sold by Alltech Inc. under the name of Bio-Mos®, was proved (in more than 600 feeding trials) to have a positive impact on the immune system of livestock (Baurhoo et al., 2009; Yang et al., 2008; Rosen et al., 2007; Jacques & Newman, 1994; Morrison et al., 2010) fish (Dimitroglu et al., 2009), and companion animals (Swanson et al., 2002) when added to feed at the rate of 0.5-2.0 kg/ton. Bio-Mos® contains ~17% of α-mannan, which is possibly its bioactive component. An improved form of Bio-Mos® is marketed by Alltech Inc. under the name ActigenTM. This improved product contains 30% α-mannan and is manufactured, by mixing yeast cell wall with a mannan-rich fraction. ActigenTM product is four times more active than Bio-Mos® (~13% of it is soluble and therefore, better available to interaction with microbes and animal gut) and its application rate of 250-500 g/ton approaches that of antibiotics. Alpha-Dmannans have a "brush like" structure built out of α-(1→2)- and α-(1→3)-D-mannopyranose branches, 1 to 5 rings long (brush hair), which are attached to a ~120-ring-long α-(1→6)-Dmannopyranose chain, the brush handle (Vinogradov et al., 1998). This structure creates a specific combination of various functionalities that also involve protein conjugates. It can fit with various receptors present on the walls of animal digestive tracts (Mansour & Levitz, 2003) and with the receptors on the membranes of pathogenic bacteria (Wellens et al., 2008). Alpha-D-mannan/protein-conjugates are involved in interactions with animal immune systems and as a result enhance immune system activity (Wismar et al., 2010) and contribute to animal antioxidant and antimutagenic defenses (Krizkova et al., 2006). Immune system strength, blockage of bacterial adhesion to the gut and modification of the gut structure contributes to improved survival and better growth in young animals. The second major yeast cell wall polysaccharide, (1→3)(1→6)-β-D-glucan, might also contribute to yeast cell wall biological properties (section 5 of this chapter), but its major role in animal nutrition is its ability to bind mycotoxins and detoxify animal feed. The (1→3)(1→6)-β-D-glucan is a part of the cell wall's "triple helix tridimensional structure, with spring-like mechanical properties, responsible for yeast cell wall strength and ability to absorb toxins" (Yannikouris et al., 2004) (see section 2, this chapter). Toxins occurring in plant-derived animal feed belong to one of two groups. Mycotoxins (the first group) are the by-products of the secondary metabolism of pathogenic fungi (Bennet & Klich, 2003), whereas chemical toxins (the second group) are incorporated into plant tissue as a result of plants metabolizing agrochemicals from the soil or water used for irrigation (McLean & Bledsoe, 1992). Some of the most problematic mycotoxins in causing human or animal diseases (i.e., aflatoxin, citrinin, ergot alkaloids, fumonisins, ochratoxin A, patulin, trichothecenes and zeralenone; Smith et al., 1995) can be absorbed by yeast cell wall β-glucans (Yiannikouris et al., 2004; Yannikouris et al., 2006). A mixture of cell wall β-glucan with clay (bentonite) sold as Mycosorb® by Alltech Inc. offers a spectrum of mycotoxin absorption superior to that of yeast cell wall glucans alone and also absorbs heavy metals (Brady et al., 1994). As in the case of Bio-Mos® a multitude of feeding trials have demonstrated the efficacy of Mycosorb® as an animal feed detoxifier using companion animals (Leung et al., 2006), horses (Raymond et al., 2003), pigs (Kogan & Kohler, 2007) and poultry (Dvorska et al., 2003; Karaman et al., 2005; Valarezo et al., 1998).

The whole yeast *Saccharomyces cerevisiae* grown in a medium containing inorganic selenium (Demirici & Pometto, 1999; Demirici et al., 1999; Mapelli et al., 2011) is used to produce yet another yeast-based human/animal nutritional supplement SelPlex® (Alltech Inc.). To maintain healthy metabolism, the human body requires 17 µg of selenium a day. Dietary selenium is used to synthesize selenocysteine (in liver) and is then incorporated into more than 25 Se-containing enzymes that play important roles in body's defense against free radical species (Tapiero et al., (2003) and in many other cellular processes including the generation of energy in mitochondria (Rayman, 2000). Because yeast does not possess genes that control selenium metabolism (Rodrigo, 2002), selenium, which has chemical properties extremely similar to sulfur, is metabolized in the same manner as sulfur and randomly incorporated into yeast cytosol, small-molecules like Se-glutathione, Se-adenosylhomocysteine (Uden et al. 2003) and proteins as Se-methionine (Tastet et al., 2008). Unlike yeast, mushrooms can metabolize selenium and can accumulate large quantities of it (Turlo et al., 2007) in the form of selenomethionine and selenocysteine. Animal feeding studies clearly showed that SelPlex® is not just a selenium source, but it also carries a variety of beneficial effects such as increased animal fertility (Rayman, 2000) or improved animal immune system activity (Rayman, 2000). Additionally, feeding SelPlex® to mice has been shown to delay the development of brain tumors from malignant human cancer cells implanted in mice brains (Toborek, 2011) and can significantly limit the deposition of Aβ amyloid plaques in APP/PS1 mouse brains that carry human Alzheimer's disease genes (Lovell et al., 2009). Only some of these effects can be promoted by regular yeast cell wall preparations. However separating the bioactivities caused by the selenomethionine-containing yeast proteins from the activity caused by the polysaccharide parts of selenized yeast cell walls is difficult. Comparison of genomic activity in tissues taken from animals fed with SelPlex® to those fed with Bio-Mos® spiked with selenomethionine indicates very large differences in the regulation of multiple groups of genes for both treatments (Kwiatkowski et al., 2011), which may indicate that the bioactivity of Sel-Plex® involves cooperation between the selenoprotein and the polysaccharide components of selenized yeast cell walls.

Indeed, selected extracts from selenized yeast/yeast cell walls (Kwiatkowski et al., 2011) show potential as future, possible treatments of diseases such as type 2 diabetes, cancer and Alzheimer's.
