Koji fermentation based on extracellular peptidases of <Emphasis Type="Italic">Flammulina velutipes</Emphasis>

A total of 28 basidiomycetes and 16 substrates were investigated to detect extracellular peptidases for the hydrolysis of wheat gluten as a complex Koji substrate. In a first screening, basidiomycetes were grown in submerged cultures containing gluten as only carbon and nitrogen source. Growth rate, protein concentration and peptidase activity were monitored. Flammulina velutipes, Armillaria mellea, Trametes versicolor, Meripilus giganteus, Fomitopsis pinicola, Phanerochaete chrysosporium and Hericium erinaceus showed high peptidase activity. To evaluate the effects of complex substrates on peptidase generation, a set of experiments was performed using surface cultures of Flammulina velutipes. This fungus grew well on many of the substrates and yielded a maximum of over 160.000 arbitrary U mL⁻¹ on the surface liquid released from gluten pellets. Gelatin zymography visualized a complex mixture of secreted peptidases. Highest peptidase yield was obtained from a koji-type culture: A maximum of 63% of the gluten was converted to free amino acids in 14 days by peptidase activities in the range of 80,000–90,000 aU mL⁻¹. The dark brown hydrolysate smelled and tasted like meat broth with no detectable bitterness.