Hydroperoxides and n-hexanal of soymilk made at different temperatures in the soybean grinding process were investigated. Both hydroperoxides and n-hexanal showed maximum amounts at 30°C, 37.78 mol/g, and 1.94 mg/g, respectively. However, at 3°C and 80°C, amounts of hydroperoxides were about half of that at 30°C. N-hexanal showed high correlation with hydroperoxides except for at 80°C. It suggests that controlling the grinding temperature is effective to reduce hydroperoxidation and off-flavor content. Protein solubility, an important index of soymilk, was decreased as the temperature increased. Grinding soybeans at low temperature is considered an economical method to produce soymilk having less off-flavor and high protein.

A thermophilic fungus, K11014, newly derived from a compost was selected on the basis of protease productivity as the only one of 81 isolates to produce high levels of acid protease. The fungus was named Penicillium duponti K1014 based on taxonomical studies. It grew in the temperature range of 28 to 58 C, and the optimum was 45 to 50 C. These temperature characteristics showed that the fungus was the most strongly thermophilic of all the fungi next to Humicola lanuginosa. When P. duponti K1014 was grown on moistened wheat bran, maximal accumulation of acid protease occurred after 2 days at 45 to 50 C. The addition of ammonium salts, but not nitrate, was effective for the production of the acid protease. The acid protease of P. duponti K1014 was stable at 60 C for 1 hr and retained more than 65% of original activity after the treatment for 1 hr at 70 C at pH 4.7. This thermal property was different from those of the ordinary acid proteases, indicating that the enzyme is a thermostable protein.

A purified acid protease from a true thermophilic fungus, Penicillium duponti K1014, was most active at pH 2.5 for milk casein and at pH 3.0 for hemoglobin. The enzyme was stable at a pH range of 2.5 to 6.0 at 30 C for 20 h. The acid protease retained full activity after 1 h at 60 C at a pH range between 3.5 and 5.5. At the most stable pH of 4.5, more than 65% of its activity remained after heat treatment for 1 h at 70 C. These thermal properties show the enzyme as a thermophilic protein. The enzyme activity was strongly inhibited by sodium lauryl sulfate and oxidizing reagents such as potassium permanganate and N-bromosuccinimide. No inhibition was caused by chelating reagents, potato inhibitor, and those reagents which convert sulfhydryl groups to mercaptides. Reducing reagents showed an activating effect. The enzyme showed the trypsinogen-activating property at an acidic pH range; optimal trypsinogen activation was obtained at a pH of approximately 3.0. The isoelectric point of the enzyme was estimated to be pH 3.89 by disk electrofocusing. By using gel filtration, an approximate value of 41,000 was estimated for the molecular weight.

A thermophilic fungus, Penicillium duponti K1014, produced an acid protease in a submerged culture. Maximum enzyme production, in a 30-liter fermentor at a preselected optimum growth temperature of 50 C, occurred after 3 days. The productivity of the enzyme was associated with changes of viscosity and pH of culture broth during the growth. The acid protease was isolated from the culture filtrates and was purified ninefold by alcohol precipitation, column chromatography on O-(diethylaminoethyl)cellulose, batchwise treatment with O-(carboxymethyl)cellulose, and by gel filtration. The purified enzyme was homogeneous on disk electrophoresis and sedimentation analysis. The sedimentation constant S20.w, calculated at a concentration of 0.64%, was 3.98S.

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