Jan. 7. 1918 Effect of Time of Digestion on Hydrolysis of Casein 3 



Five lo-gm. portions of Hammarsten's casein were weighed out and 

 transferred to five i -liter round -bottom Jena flasks. Fifty gm, of 

 cornstarch were then weighed out and added to each of the flasks except 

 the first, which contained casein alone. Three hundred c. c. of 20 per 

 cent hydrochloric acid, specific gravity i.ii, were added to each flask. 

 All the flasks were then heated on the water bath, with frequent shak- 

 ings, for about two hours. The object of this preliminary heating on 

 the water bath was to liquefy the starch-casein mixtures, which had 

 gelatinized upon the addition of the hydrochloric acid. After the starch 

 had become liquid all the flasks were removed and attached to reflux 

 condensers and heated to a gentle boil. 



Experiments i and 2 were allowed to digest for 12 hours, No. 3 for 15 

 hours. No. 4 for 24 hours, and No. 5 for 48 hours, each being cut out at 

 the expiration of its time interval. 



After each of the experiments had stood at room temperature for six 

 or eight hours, they were filtered through paper on a Buchner funnel 

 and washed practically free of chlorids with hot water. There was no 

 insoluble residue remaining on the filter from the casein digestion. There 

 were rather large insoluble carbonaceous residues remaining from each 

 of the casein-starch mixtures. Each of these was dried at 100° C, 

 bottled, and set aside for further investigation as to their nitrogen 

 content. 



The filtrates in each of the experiments were concentrated separately 

 under reduced pressure until practically all of the excess of hydrochloric 

 acid was removed. The residues were taken up in water and run through 

 filters into separate flasks of 250-c. c. capacity. After the filters were 

 washed thoroughly, the contents of each flask were brought up to the 

 mark with water, and duplicate analyses were carried out by the Van 

 Slyke method on aliquots from each of these hydrolyzed solutions. 

 The results obtained are shown in Table I. 



From the data in Table I showing the average results obtained upon 

 casein alone and upon definite mixtures of starch and casein digested at 

 different intervals of time the following observations may be made. 



In all of the experiments there is but slight variation in the ammonia 

 determinations^ ; the maximum result is obtained in the 48-hour digestion. 

 The increase in this case is in all probability owing to the change of some 

 of the amino groups to ammonia compounds, which indicates over- 

 digestion. 



The results for the humin determinations show a diminution in the 

 1 5-, 24-, and 48-hour digestions over those of the 12-hour digestions. 

 However, the humin determination in the 12-hour digestion of the 

 starch-casein mixture agrees well with the humin results obtained on 

 casein alone. 



' Previous to the amraonia determinations the acidity of the hydrolyte, in terms of the calcium-hydrate 

 suspension, was determined by titration, with phenolphthalein as the indicator. A slight excess of the 

 calcium-hydrate suspension aoove tne amount necessary to neutralize the acid was always added. 



