**5. β-(1**→**3)(1**→**6)-D-glucan as valuable by product from yeast fermentation**

56 The Complex World of Polysaccharides

2005; Valarezo et al., 1998).

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.,

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). The commercial source from which the bulk yeast cell wall polysaccharides (including βglucan) are produced uses the same strains of yeast as are used in fuel alcohol production. Le Saffre/ADM, Lallemand, Enzyme Development (New York) and DSM Life Sciences (Delft) are the largest suppliers of yeast for fuel ethanol producers in the United States and the European Union. Major factors that affect yeast cell wall composition include yeast strain (Hahn-Hagerdal et al., 2005), growth conditions (growth medium, temperature, osmotic pressure, toxic metabolites) and the time of harvesting (Aguilar-Uscanaga & Francois, 2003; Klis et al., 2002; Klis et al., 2006). Fuel alcohol fermentation is a high stress process (Devantier et al., 2005) and the cell walls of the yeast collected as its byproduct contain a high amount of β-glucans (Basso et al., 2008; Knauf & Kraus, 2006; Jones & Ingledew, 1994). In general, yeast strains of *Saccharomyces cerevisiae* that are used in baking (baker's yeast) have a higher β-glucan-to-α-mannan ratio than those that are used for alcohol fermentation (brewer's yeast), therefore it is advantageous to use pure baker's yeast for producing high quality (1→3)(1→6)-β-D-glucan for medicinal applications (Kim et al., 2007). The process of separating various yeast components has been heavily patented. However, the differences in the technologies are minor and in principle do not differ from the methodology described by Manners and Fleet (1976). The process starts from autolysing yeast cells at a temperature between 45oC and 65oC at slightly acidic pH, to release yeast cell walls that are insoluble and denser than the cytoplasmic contents and can be separated by centrifugation (Wheatcroft et al., 2002). These steps can be followed by incubating the yeast cell walls with alkaline protease at a pH of 9 to 10 to solubilize mannans and leave behind insoluble β-glucan (Zapata et al, 2008), which can then be physically separated from the liquid fraction by centrifugation and subsequent washing (Sedmak, 2006). Additional enzymes, like glucoamylase and lipase, can be used to hydrolyze α-glucan from α β-glucan, which is still present in the cell wall material and to solubilize the residue lipids from cell membranes. The final step of β-glucan production is a spray-drying that produces a whiteto-maroon colored powder that does not carry any taste or aroma and is useful for feed and food applications. Further alkaline and acidic treatments of the food-grade β-glucan (Kelly,

2001) yields high purity (98.5% β-glucan, <0.1% mannan, 0.4% α-glucan, 0.3% protein, 0.2% chitin) microparticulate β-glucan with reduced molecular weight (from ~1-3 MDa to ~150 kDa) that is much more easily absorbed by the digestive tract and shows improved activity compared with food-grade products containing only ~65% β-glucan. Even further hydrolysis produces soluble yeast β-glucan (Jamas et al., 1998; Lee et al., 2001) that still retains most of the particular β-glucan bioactivities (Janusz et al., 1986; Wakshull et al., 1999).

Yeast *Saccharomyces cerevisiae*, its cell wall and products of its fractionation are generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA, 1997), and they can be legally used as food ingredients but not as food additives. The European Food Safety Authority (EFSA) issued an opinion that yeast β-glucans are a "safe food ingredient" (EFSA, 2011) that can be used as a "food supplement" up to 375 mg/day and in foods for "particular nutritional uses" at dose levels up to 600 mg/day. (The uses of yeast cell wall as an animal feed ingredient were discussed in section 4 of this chapter).

Food-grade yeast β-glucans such as BetaRight® and WGP® (Biothera, Inc.) are used as ingredients in baked foods, beverages, ceral, yogurt, fruit juices, chocolate and as food thickeners in salad dressings, ice cream, mayonnaise, sauces and cheese. The majority of these applications have been patented (Zechner-Krpan et al., 2009; Thammakiti et al., 2004) and a critical review of 300 patented applications is available (Laroche & Michaud, 2007). Yeast β-glucans improve food rheological properties, gelling, water and oil-holding properties, without impacting its taste or odor (Petravic-Tominac et al., 2011). Beta-glucans also add health benefits (Laroche & Michaud, 2007) like antioxidative, bacteriostatic and immunostimmulating activities. Cosmetic products used in skin treatment contain yeast βglucans as moisturizing and moisture-retaining components that also provide a proper moistening feeling. Because of its emulsion-stabilizing effects, pleasant texture and antioxidant activity yeast β-glucans can prevent skin injuries caused by solar radiation and therefore are used in sun-screens, oils and gels (Michiko & Yutaka, 2007). Deodorants containing yeast β-glucans have proved to be useful in oral preparations, mouthwashes and diapers (Michiko et al., 2005). Acid-treated cell walls (AYC) can be used as new binders in pharmaceutical formulations and, when mixed with traditional fillers like hydroxypropylcellulose or polyvinylpyrrolidone, yield harder pills with very short (~2 min) dissolution times (Yusa et al., 2002). Its adhesive and biological properties can be also utilized in producing coating for surgical instruments (Klein, 2003) and in the manufacture of packaging for the food industry (Cope, 1987). Its antibacterial and antiviral properties have found application in the control of plant pests (Kitagawa, 2007) and viral invasions (Slovakova et al., 1997).
