Lentinula Edodes Biotechnology – from Lentinan to Lectins

230 V.E. NIKITINA et al.: Lentinula edodes Biotechnology, Food Technol. Biotechnol. 45 (3) 230–237 (2007) ISSN 1330-9862 review (FTB-1921) Lentinula edodes Biotechnology – From Lentinan to Lectins Valentina E. Nikitina1, Olga M. Tsivileva1*, Alexei N. Pankratov2 and Nikolai A. Bychkov1 1Laboratory of Microbiology and Mycology, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 13 Entuziastov Avenue, 410049 Saratov, Russia 2Department of Chemistry, N. G. Chernyshevskii Saratov State University, 83 Astrakhanskaya Street, 410012 Saratov, Russia Received: September 27, 2006 Revised version: March 20, 2007 Accepted: May 18, 2007 Summary Lentinula edodes was the first medicinal macrofungus to enter the realm of modern biotechnology. The present paper briefly reviews the history of the modern biotechnology of this mushroom starting with the production of the polysaccharide preparation lentinan, and ending with an overview of our own work regarding the production of lectins. Our work with lectins has involved studies of the effect of initial pH, carbon and nitrogen sources and the C:N ratio on lectin production in both the mycelium and culture medium. We have shown that lectin activity is related to morphological development, with the activity being highest in extracts of the pigmented mycelial films that precede fruiting body production. Key words: submerged culture, Lentinula, lectins of higher fungi, brown mycelial film, molecular structure, quantum chemical study both mushroom crops and mushroom derivatives should Introduction have a positive global impact on long-term food nutri- tion, health care, environmental conservation and regen- The production volume of mushrooms has increased eration, and economic and social change (4). dramatically over the last several decades. For example, the annual production of the popular Agaricus bisporus Whenever mushroom derivatives are the desired pro- has increased from 30 000 tonnes in 1970 to 300 000 ton- ducts, the disadvantages that traditional fruiting body nes in 2001 (1). production suffers from are well known: relatively long Mushrooms do not merely constitute a highly nutri- cultivation times and the possibility of variations in pro- tious source of food. More recently, attention has focused duct quality due to unavoidable variations in growing on a second area of exploitation, following the discovery conditions. The advantage of submerged mushroom cul- that many of these fungi produce a range of metabolites ture for the production of mushroom derivatives is clear: of great interest to the pharmaceutical and food indus- mycelia formed by growing pure strains in submerged tries (2,3). As a result, the present-day mushroom indus- culture under controlled conditions not only have a con- try is based on two main components: the application of sistent composition, but are also safer (5,6). traditional, although modernized, techniques for the One of the first macrofungi to be cultivated at large production of fruiting bodies (i.e. mushrooms them- scale in submerged culture was the basidiomycete Len- selves) and the application of modern biotechnological tinula edodes (Berk.) Pegler, otherwise known as shiitake. techniques to produce mushroom derivatives such as Over the last 30 years the morphology, life cycles and nutriceuticals and dietary supplements (4). Products from genetics of shiitake have come under scrutiny and laid *Corresponding author; Phone: ++7 8452 970 444; Fax: ++7 8452 970 383; E-mail: [email protected] V.E. NIKITINA et al.: Lentinula edodes Biotechnology, Food Technol. Biotechnol. 45 (3) 230–237 (2007) 231 the foundations for current methods of selection and cul- sion at this time is that the immunopotentiating activity tivation of this mushroom (7). The current paper briefly of (1®3)-b-glucans depends on a helical conformation and reviews this history, and shows that we still have impor- on the presence of hydrophilic groups located on the tant gaps in our knowledge of the biochemical aspects outside surface of the helix. of L. edodes development and cultivation, especially in Polysaccharides from mushrooms do not attack can- liquid media. It then goes on to describe some of our re- cer cells directly, but produce their antitumor effects by cent work with submerged culture of this mushroom for activating the immune responses of the host, stimulating the production of lectins. It does not cover traditional natural killer cells, T cells, B cells, and macrophage-de- techniques for fruiting body production, since these top- pendent immune system responses (30). The immuno- ics have already been well reviewed (8–23). modulating action of mushroom polysaccharides is especially valuable as a means of prophylaxis (a mild and non-invasive form of treatment), prevention of metasta- Biosynthesis of Extracellular Polysaccharides tic tumors, and as a co-treatment with chemotherapy. and Other Medicinal Substances The commercial polysaccharide »lentinan« from the shiitake mushroom offers the most clinical evidence for The medicinal properties of substances occurring in antitumor activity (26). In fact, lentinan is one of the first higher basidiomycetes have become a subject of thorough three major drugs developed from medicinal mushrooms, investigations (24). This is especially true for the shiitake b mushroom, the medicinal and therapeutic values of which all three being -glucans. Lentinan is produced in sub- have been recognized for many decades (25). Mush- merged, agitated liquid cultures, giving biomass concen- rooms comprise a vast and yet largely untapped source trations between 7 and 8 g/L, with the lentinan yield of powerful new pharmaceutical products, particularly being greatest at pH=5 (31). Polysaccharides with simi- (and most importantly for modern medicine) represent- lar infrared spectra to those obtained from the fruiting ing a source of a large number of polysaccharides with bodies of L. edodes have been extracted from biomass and antitumor and immunostimulating properties (26). Their from the culture liquid itself. There appears to be some practical application depends not only on their unique difference in the molecular composition of the intra- and properties but also on biotechnological availability. Iso- extracellular lentinan. lation and purification of polysaccharides from mush- Lentinan produced extracellularly is much more ef- room material (i.e. intracellular polysaccharides from fective in stimulating antibody production in mice than mycelium and extracellular ones from culture broth) is that extracted either from fruiting bodies or from the bio- relatively simple and straightforward, and can be car- mass produced by fermentation methods. However, more ried out with minimal effort (27,28). recently, lentinan has been extracted from L. edodes my- The biochemical mechanisms that mediate the bio- celium via a new cost-effective procedure that resulted logical activity of polysaccharides are still not clearly in high purity (88 %) and yield (32). Unlike previous studies, in which lentinan was given parenterally, in this understood. What is obvious is that the (1®3)-b-glu- study it was administered orally. It was shown that the can backbone is essential, and that the most active poly- antitumor efficacy was still expressed through this route mers have degrees of branching (DB) between 0.20 and of administration. 0.33 (i.e. a branch every three to five backbone residues). (1®3)-b-Glucans that have b-glucopyranosyl units at- More and more pharmacologically active metaboli- tached by (1®6) linkages as single unit branches en- tes from mushrooms are being isolated and tested. For hance the immune system systemically (29). This en- example, edible mushrooms including L. edodes have been hancement results in antitumor, antibacterial, antiviral, reported to have anti-hypertensive effects (33). As an- anticoagulatory and wound healing activities. other example, the mycelium-free broth of L. edodes grown in submerged liquid culture was bacteriostatic against Strep- The currently available information about which poly- tococcus pyogenes, Staphylococcus aureus and Bacillus me- saccharide properties are essential for immunocompetence gaterium, but had no antifungal activity against Candida is not necessarily contradictive, although the conditions albicans (34). The substance responsible for the activity that have been used for evaluating these properties are was heat-stable, could be extracted with chloroform and not uniform. As a result, some data suggest that triple had a molecular mass under 10 000. These characteris- helical structures formed from high molecular mass poly- tics suggested that the component might be lenthionine, mers are possibly important for immunopotentiating ac- an antibacterial and antifungal sulphur-containing com- tivity, while other data suggest that the activity is inde- pound. The culture fluid was less toxic to human tissue pendent of any specifically ordered structure. Interpreta- culture cells than to microbes. The antibacterial activity tion of these results is complicated by the fact that there and the toxicity to human cells did not reside in the same are still other data which indicate that it is the distribu- component. tion of the branch units along the backbone chain that is the main determinant of the activity. Futhermore, there are data that indicate both that b-glucopyranosyl units Aflatoxin-Inhibiting Effect of Lentinula edodes are required for immunopotentiating

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