Nov. 26, 1917 
Enzyms of Milk and Butter 
443 
proteolytic enzym of milk is not of bacterial origin, as Olson (14) recently 
reported. 
(/) Amount of 1 gaeactase contained in butter. —The butter 
used in the experiments represented both good and bad qualities. The 
“fresh dairy” butter was made from the cream obtained from the milk 
of the University Farm herd, and was soured spontaneously without 
pasteurization. The “fresh creamery” butter was made in a cooper¬ 
ative creamery from sweet pasteurized cream ripened by a commercial 
starter. Both of these butters were of extra-fine quality. The “stored 
dairy” butter was the same as the “fresh dairy,” except that it had 
been held in cold storage for eight months. The “fresh centralized” 
butter was made from the sour cream just as it was received by a central 
creamery. The last two butters mentioned were of poor quality. The 
“stored centralized” butter was made in the same way as the “fresh 
centralized,” but had been held eight months in cold storage. It was of 
extremely poor quality. 
No effort was made to obtain a pure enzym extract from the butter. 
A known weight, usually 400 gm., of butter was melted at 45 0 C. in two 
long glass tubes about an inch in diameter. After the separation was 
complete the clear fat was hardened by immersing the tubes in cold 
water, and the curd solution was then washed out. If an excess of fat 
remained in the extract, it was removed by rewarming and centrifuging. 
This extract was then dialyzed at a temperature never exceeding i3°C., 
in a parchment dialyzer, until free of sodium chlorid, the last six or eight 
hours of dialysis being with distilled water. This curd was made up to a 
given volume and used at once in the various tests. 
The acetone-ether method of obtaining a fat-free, dry powder was 
also tried, but so high a percentage of sodium chlorid was left in the 
powder that dialysis was necessary. Consequently this method had no 
advantages over the other, and the enzyms probably would have been 
weakened by the precipitation. 
The casein was precipitated by the optional official method (26, 
p. 118), rather than the official method because filtering was often more 
rapid, the volume to be filtered much less, and a clear filtrate more easily 
obtained. (Thirteen analyses of chloroformed-skim milk and butter- 
curd extracts gave an average of 0.00104 gm. more nitrogen in the form 
of casein by the use of alum as the precipitant; hence, the methods should 
not be used interchangeably.) A clear filtrate was more easily obtained 
and an excess of substrate assured by the addition of boiled skim milk 
to the curd solutions. In a few cases the filtering had to be done on a 
“Buchner funnel” through three filter papers and repeated 10 to 20 
times to obtain a perfectly clear filtrate. Bacterial action was prevented 
by 1 per cent chloroform instead of 0.5 per cent because the extracts 
contained considerable fat (Table VI). 
