A Potent fungus for producing milk clotting enzyme was selected out from some 300 strains of soil fungi and was identified as Mucor racemosus Fres. The ratio of milk clotting activity (MCA)-to-proteolytic activity (PA) of milk clotting enzyme is an important index for estimating it as a rennet substitute. However, the MCA/PA ratio of the crude enzyme derived from the fungus was lower than that of calf rennet or Mucor pusillus rennet. Mutants of the fungus were induced by successive N-methyl-N' nitro-Nnitrosoguanidine (NTG) treatment or ultraviolet (UV) irradiation in an attempt to increase the MCA/PA ratio. Some stable mutants that produced enzymes with a high MCA/PA ratio were selected out from the NTG-treated or UV irradiated strains. The MCA/PA ratio of the crude enzyme from the fourth generation NTG mutant No. 51 and the fourth generation UV irradiated mutant No. 54 was 1.5_??_1.7 times higher than the ratio of the crude enzyme from the parent strain and nearly equal to that of Mucor pusillus rennet.
The MCA/PA ratios of calf rennet and the crude enzymes from the parent strain and its mutants increased by using 0.44M TCA instead of TCA mixture as the agent for PA assay, but the MCA/PA ratio of the crude enzymes from Mucor pusillus, Irpex lacteus and Bacillus polymyxa were almost uninfluenced by this change in mixing. The microbiological characteristics of these two mutants were similar to those of the parent strain, except for the height of the turf, the temperature range for growth and utilization of carbohydrates. The maximum enzyme production of these two mutants by solid culture on wheat bran medium at 24°C reached about 6500 soxhlet units per gram of wheat bran. The enzyme was quickly produced on wheat bran medium, but after the production reached maximum, the enzyme activity decreased sharply.

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Mucor racemosus No. 50 and its mutants produced milk clotting enzymes in high yield on solid wheat bran media. The enzyme was extracted with water and precipitated by salting out with ammonium sulfate. Some enzymological properties of these crude enzymes (SR enzymes) were studied and compared with those of a calf rennet (HR) and Mucor pusillus rennet (MR). SR enzymes were acid proteases with an optimum pH of 3.0 for casein digestion. The differences in enzymatic properties between the parent strain and its mutants were in the ratio of milk clotting activity (MCA)-to-proteolytic activity (PA) and certain other properties, such as pH optima for hemoglobin digestion and pH-casein digestion curves. The increase in MCA/PA ratio of the crude enzyme from the mutant was due to PA being lower in the mutant than in the parent strain at a pH range from 5.5 to 7.0.
The crude enzymes from the mutants had a higher in the MCA/PA ratio than MR and other microbial rennets, using 10% reconstituted skim milk (RSM) containing 1/1000M CaCl₂ (approximately equivalent to quantities of CaCl₂ used for cheese making) as a substrate for determining MCA, but were nearly equal to MR, using 10% RSM containing 1/100M CaCl₂.
SR enzymes were less heat stable than HR and MR, and less resistant against pH change than MR. The optimal temperature for MCA was around 56°C and 46°C, on a substrate of 10% RSM containing 1/100M CaCl₂ and 1/1000M CaCl₂, respectively, Milk clotting activities of SR enzymes were less affected by the Ca ion concentration and pH of milk than those of MR and HR.

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Irradiated D-fructose solution exhibited marked enhancement in browning reaction with amino acid, and this effect depended on some radiolytic products of fructose.
The radiolytic products formed in solution under aerated and anaerobic conditions were fractionated by column chromatography on Sephadex G-10, LH-20 and ion-exchange resin and by TLC on silica gel. The browning activity of each fraction was determined by reaction with glycine. The browning active products in the fraction were identified by GLC, GC-MS and TLC; they were: 6-deoxy-D-threo-2, 5-hexodiulose, D-threo-2, 5-hexodiulose, D-arabinohexosulose and 4-deoxy-L-glycero-2, 5-hexodiulose from irradiated solution under anaerobic conditions and D-threo-2, 5-hexodiulose and D-arabinohexosulose from solution under aerated conditions. The browning reactions of the model compounds with glycine also indicated that D-threo-2, 5-hexodiulose and 6-deoxy-D-threo-2, 5-hexodiulose were more active than D-arabinohexosulose and 3-deoxy-D-erythrohexosulose.

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Essential oils were obtained from Rudbeckia laciniata L. in ca. 0.03% yield by steam distillation. The plants were collected at Gifu Shiratori in August, 1978, and at Gifu Takayama in August, 1979. The essential oils were chromatographed on activated alumina, and separated into five fractions; n-hexane, benzene, diethyl ether, ethyl acetate and methanol fraction. This essential oil was composed of terpene, aliphatic and aromatic compounds, in which the main components were α-pinene, limonene, β-phellandrene and bornyl acetate.

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