764 Journal of Agricultural Research vol. xx,No. IO 



After the flasks had been heated for 1 5 minutes the contents were filtered 

 through a fine quality of absorbent cotton to remove the mycelium and 

 sand. Filter paper was first tried but was finally rejected in favor of the 

 cotton for two reasons: (1) The solution filtered slowly, thereby intro- 

 ducing considerable error as a result of evaporation; (2) it removed 

 much of the nonhydrolyzed starch. After the filtrate cooled, the reducing 

 sugars were determined volumetrically, according to the method of 

 Clark (8). This is a quick and accurate method for the determination 

 of small amounts of reducing sugars by titrating the reduced copper 

 without removing it from the residual copper solution. 



The results of starch hydrolysis set forth in the discussion of the 

 following experimental data are expressed in milligrams of reducing 

 sugars in a given volume of solution or in total reducing sugars formed. 

 The results are expressed mostly in milligrams per 10 cc, because 10 cc. 

 of solution are usually employed in making the titrations. If the quan- 

 tity of reducing sugars in 10 cc. of solution is known, the total reduc- 

 tion or that portion of the starch remaining nonhydrolyzed can be 

 calculated. 



It is evident from the method employed that no account is taken of 

 products intermediate between the starch and reducing sugars. It is 

 likely that such products, for example dextrins, are formed in all cases, 

 but the determination of the reducing sugar meets the requirements of 

 the problem in hand, which has for its object mainly to show that a 

 vigorous starch-splitting enzym is formed by Rhizopus tritici, and also 

 some of the conditions upon which the production of this enzym -depends 

 and how certain environmental factors may influence its activity. 



Various modifications of these methods were used in certain of the 

 experiments, but such changes in the methods required to meet the 

 needs of the experiments will be explained in sufficient detail when the 

 results of the experiments are presented and discussed. 



It was shown by Dox (9) that a considerable autolysis of the fungus 

 mycelium actually takes place. In some enzym experiments where 

 hydrolysis is measured by the amount of reducing sugars formed, a 

 considerable error is likely to be introduced if a correction is not made 

 for the autolysis of the mycelium itself. A number of tests have shown 

 that the amount of autolysis produced from 0.25 gm. of mycelium sus- 

 pended in 50 cc. of distilled water varies from 1.20 to 7.39 mgm. per 

 10 cc, with an average of 6.38 mgm. Where a considerable amount 

 of reduction of the starch is involved, this amount would not introduce 

 a very considerable error. On the other hand, where the total hydroly- 

 sis is small a considerable error in the final results might be introduced. 

 In all experiments, except where the results would not be influenced 

 one way or the other, the autolysis of the mold was determined and 

 deducted from the total reducing sugars formed in the system. 



