Development of mushrooms for thrombosis prevention by protoplast fusion

With thrombosis a major cause of death in Japan and the Western world, thro mbin-inhibitory agents that constrain the formation of fibrin are sought. W e screened for basidiomycetes showing anti-thrombin activity and isolated L aetiporus sulphureus. However, it was difficult to cultivate and its form w as not satisfactory. We therefore used protoplast fusion between L. sulphur eus and the commonly cultivated basidiomycete Hypsizygus marmoreaus to obta in cultivable basidiomycetes that produced an anti-thrombin substance. For the protoplast fusion of L. sulphureus and H. marmoreaus, the protoplast co ncentration, alternating electric field intensity, dielectrophoresis durati on, and field pulse intensity used were of 1 x 10(7) protoplasts/ml, 100 V/ cm.1MHz, 60 s, and 8 kV/cm, respectively. The number of regenerated colonie s obtained was 4961, from which 43 strains were selected for electrophoreti c analysis. Four of the fusants were found to have a band from each parent in isozyme patterns obtained using their crude extract. The fruiting bodies of the fusants were very similar to those of H. marmoreaus. Crude extract from each of the fusants and from L. sulphureus showed anti-coagulative act ivity in terms of the thrombin clotting time. We thus obtained improved bas idiomycetes that produce an anti-thrombin substance, are easily cultivated, and whose form resembles H. marmoreaus, a commonly used culinary mushroom.