ENZYMES. FERMENTATION 417 



(a) Lin t tier s Method. 



In each of a series of ten clean test tubes is placed the same ^quantity of 

 soluble starch solution (10 c.c. of 2 per cent.) and then a progressively in- 

 creasing quantity of enzyme solution, thus OT c.c. in No. i, 0*2 c.c. in No. 

 2, 0-3 c.c. in No. 3, and so on. The contents are mixed and placed in a 

 water-bath at 21 for exactly i hour. 5 c.c. of Fehling's solution are now 

 placed in each tube, the tubes are heated in a boiling water-bath for 20 

 minutes and then examined. Some of the tubes in the series will show no 

 blue colour, or are faintly yellow, whilst others are still blue. The amount 

 of enzyme in the first colourless tube is that amount which will just convert the 

 fixed amount of starch into maltose in the given time. 



The diastatic power is based upono-i c.c. enzyme solution and called 100. 

 .-. if the result was between the sixth and seventh tubes the diastatic power 

 D, is 



100 



^5 x 0>I - '5-55. 



Generally i -5 is deducted as due to reducing sugars in the extract. The 

 extract is diluted with an equal volume of water, if D is very high. 

 It is generally necessary to repeat the experiment once or twice. 



(b) Lings Method. 



3 c.c. of the diastase extnict are added to 100 c.C. of 2 per cent, soluble 

 starch solution in a 200 c.c. measuring flask heated to 21. It is allowed to 

 act for i hour at this temperature. 10 c.c. of *iN caustic alkali are added 

 to stop the action; the solution is cooled to 15 -5, made up to 200 c.c. and 

 well mixed. The amount of reducing sugar is estimated against 5 c.c. 

 Fehling's solution heated to boiling over a naked flame, the solution being 

 added slowly, 5 c.c. at a time, and kept boiling until the reduction is com- 

 plete as ascertained by Ling's indicator. The result is calculated from 



D loco 



xy 



where D is the diastatic activity, x c.c. of malt extract in 100 c.c. of 

 diluted solution, jy = c.c. of liquid required to reduce the Fehling's solution. 



It is not accurate for values of D above 50 so that less malt extract 

 (2 c.c. or i c.c.) must be taken and the meas'irement repeated. 



(c) Roberts' Method. 



Though not so accurate, this method is the most rapid to carry 

 out. Here the time taken to effect the change of I per cent, starch 

 solution into achroodextrin is measured. The stage at which no 

 colour is given by iodine solution, i.e. when the last traces of erythro- 

 dextrin have been converted into achroodextrin, is known as the 

 achromic point. The time taken to reach this point is termed the 

 " chromic period ". The time taken to reach the achromic point must 

 be between 2 and 10 minutes. The diastatic power D is the number 

 of c.c. of starch solution which can be converted by I c.c. of enzyme 

 solution in 5 minutes, or 



D . n x I 



V t 



where n = number of c.c. starch solution taken, v volume of enzyme 

 solution (dilution must be known), t = time, 5 = 5 minutes. 



