Oct.,
1920
T H E JOC.RNAL OF I-VDCSTRIAL A N D E N G I N E E R I N G C H E M I S T R Y
prevent aeration. The results of 63 such determinations are given in Table I. I n only one case was t h e difference between t h e determined and calculated Ph+ greater t h a n 0.3, and t h e mean variation is about 0.1. It will also be noted t h a t t h e wider variations occurred in t h e cases of low bicarbonate content, which is t o be expected from t h e assumptions made in t h e development of t h e equation. Somewhat larger variations are found in cases of high free carbon dioxide content t h a n in similar cases with less carbon dioxide. This is probably due t o t h e loss of carbon dioxide by aeration in t h e determination of both t h e carbon dioxide content and t h e Ph+.
From their experierlces with t h e colorimetric Ph+ determinations, t h e authors do not feel t h a t determinations can be made much more accurately t h a n 0.2 Pj2+,using open tubes and ordinary methods of transferring t h e test sample t o t h e tube. T h e effect of aeration of such unstable solutions as natural waters should amount t o this much or more. It is advisable in all cases t o make several determinations. T h e formula cannot be applied t o waters which are alkaline t o phenolphthalein. An a t t e m p t was made t o develop such a n equation, but no waters naturally alkaline t o phenolphthalein were available for checking t h e calculations. For unusual cases of this kind and cases of low bicarbonate content, it may be better t o use some more complete and more complex equation, such :is has been developed b y Prideaux.l C 0 NC L C SI 0 N S
Fairly accurate calculations of t h e H+-ion concentration of natural waters can be made from t h e simple mass law equation of t h e primary ionization of carbonic acid. Equations are developed for calculating H+-ion concentration, in which t h e carbon dioxide and bicarbonate are expressed i n t h e manner in which they are ordinarily determined. These equations are less accurate with low bicarbonate concentrations and do not apply t o waters alkaline t o phenolphthalein. X C K N 0W LEDGMEST
The authors wish t o express their appreciation of t h e assistance and advice of Prof. Victor E. Shelford, of t h e Department of Zoology, University of Illinois, who suggested t h e desirability of studying this subject , and assisted in various ways throughout t h e study. A N E W TECHNICAL M E T H O D FOR THE E S T I M A T I O N OF THE SACCHAROGENIC P O W E R OF DIASTATIC
PREPARATIONS By Kokichi Oshima TAKAMIND LABORATORY, INC., CLIFTON,N. J. Received June 1, 1920
The most practical method for t h e estimation of t h e saccharogenic power of diastatic preparations is t h a t of LintnerJ2 which is briefly outlined: 1 2
Proc. R o y . S o c . London [A], 91, 5 3 5 . 2. Drakt. Chem., 34 (1886), 386.
991
L I S T N E R METHOD
Separate volumes of 0.1,0.2, 0.3, up t o 1.0cc., of j per cent diastase solution are added t o a series of I O test tubes, each containing I O cc. of 2 per cent soluble starch solution. T h e tubes are allowed t o incubate for one hour a t constant temperature, a t t h e end of which time j cc. of Fehling’s solution are added t o each tube, t h e liquids mixed, and t h e tubes immersed in a boiling water b a t h for I O min. If, a t t h e end of t h a t period, t h e Fehling solution in t h e t u b e containing 0.1 cc. of diastase solution is just completely reduced, t h e diastatic power of t h e diastase is taken as 100. If 0 . 2 cc. reduces t h e j cc. of Fehling’s solution, t h e diastatic power is 50, etc. More exact results may be obtained, if necessary, b y taking 0.1,0.15, 0 . 2 , 0 . 2 5 cc., etc., of t h e diastase solution for a series of tubes, and determining more accurately t h e amount of digest which will just reduce 5 cc. of Fehling’s solution. T h e following formula is used for calculating t h e diastatic power of malt by t h e Lintner scale: V : 0.1 = IOO : diastatic power V is t h e volume of diastatic solution, 0.1being t h e unit quantity used t o make t h e value of I O O on t h e Lintner scale. FAULTS O F L r K T S E R hfETHoD-This method has justly been criticized by Sherman and his associates:l first, as not being accurate, since there are only ten points on t h e Lintner scale a t which an accurate determination can be made in t h e first operation; second, the probable error of t h e method increases rapidly with t h e diastatic power of t h e sample; if t h e end-point falls between t h e last tm7o tubes (0.9 and 1.0 cc.) t h e diastatic power will be between I O and 11.1, with a relatively small ‘variation;but if it falls between t h e first two tubes (0.1and 0 . 2 cc.) t h e diastatic power lies between 50 and 100, with a very large possible error. T h e Lintner method can be further criticized as n o t easy or quick enough for technical purposes, particularly when a large number of tests have t o be made as a routine in factory procedure. For example, t o test I O samples by t h e Lintner method, one has t o use I O O test tubes, and different quantities of t h e diastatic solutions have t o be tested I O O times a t t h e definite intervals of time. MODIFICATIONS OF METHOD-Ling2 modified t h e Lintner method as follows: A quantity of diastatic solution is added t o I O O cc. of 2 per cent soluble starch solution in a zoo cc. flask, and kept a t a constant temperature for one hour. At t h e end of this time, 2 0 cc. of 0 . I N sodium hydroxide are added and t h e solution is made up t o zoo cc. with distilled water. After mixing, this solution is introduced into a buret and gradually run into j cc. Fehling’s solution, diluted with a little water and kept. boiling, until t h e solution just loses its blue color. If 2 5 cc. of t h e liquid (100cc. of which correspond t o I g. of soluble starch and 1 . 5 cc. of malt extract) are required t o reduce j cc. of Fehling’s solution, t h e 1
J. A m . Chem. Soc., 32 (1910), 1075.
2
Euler’s “General Chemistry of Enzymes,” 1912, p. 290.
992
T H E J O U R N A L O F I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y Vol.
diastatic power of the sample will be
IO0
= 26.7. X I.; F r o m practical considerations, this method also has various defects, chief among which are the slowness a n d inexactness of determining t h e quantity of sugar. There is no accurate agreement with t h e Lintner scale, since, by t h e method of Ling, t h e quantity of enzymes a n d production of sugar do not give a n exact proportion; particularly is there a noticeable difference when t h e production of sugar is too much or too little. T o obviate these difficulties, Sherman and his associates proposed a method which depends upon t h e gravimetric estimation of copper oxide. T h e method i s , however, for t h a t reason too troublesome for routine factory work. PROPOSED
Strength of Extract Per cent 4 3 2 1.5 1
Zj
METHOD
I-In t h e case of a solid sample, it must be first extracted with distilled water for 3 hrs. a t room temperature. From I t o I O g. of t h e sample are usually extracted in I O O cc. of water. If t h e enzyme is too concentrated, t h e solution is further diluted with a known amount of distilled water. 2-Ten cc. of t h e extract or the diluted extract are p u t into a flask containing I O O cc. of 2 per cent soluble starch solution, which has been placed in a water bath kept a t 40' C., t h e contents of flask are mixed, and the flask is allowed t o remain in t h e b a t h for 30 min. At the end of t h a t period, I O cc. of 0 . 2 N sodium hydroxide are added a t once t o stop further enzymatic action. 3-Graduated quantities of t h e digest are then poured into a series of test tubes each containing j cc. of Fehling's solution; a n d the tubes are placed i n a boiling water bath for I O min., t h e tubes being shaken once during t h a t period. The smallest quantity of t h e digested starch solution which just reduces 5 cc. Fehling's solution is determined. It may be more convenient t o use a t first only 2 or 3 tubes containing quantities of t h e digest within wide regions of variation, so as t o determine t h e approximate concentration of the digest t o be used. The reduction of t h e Fehling solution is then carried out within t h e narrow limits of this approximate value. 4-Knowing t h e amount of digested starch solution which will just reduce j cc. of Fehling's solution, we can calculate, as shown later, t h e Lintner value. The exact value is obtained b y dividing t h e value found i n t h e table by t h e percentage of the solid or liquid sample in t h e diluted enzyme solution. CALCULATION T O LINTXER SCALE-Lintner's scale is very useful and popular; it was, therefore, found convenient t o recalculate the values obtaEned by t h e new method in terms of this scale. An extract of a culture of Aspergillus oryzae (grown on bran) and a n extract of malt were used in this investigation. The experiments were repeated numerous times so as t o verify t h e results. It was found t h a t , with a definite extract of A s p e r gillus oryzae grown on bran, tested b y t h e Lintner method a t 40' C., 0 . 2 6 2 cc. of I per cent solution reduced 5 cc. of Fehling's solution. The following results were obtained by using the new method:
12,
No.
IO
Vol. of Digest Required to Reduce 5 Cc. Fehling's Solution
CC.
3.5 4.0 5.0 5.75
7.5
I n each case, I O cc. of the particular concentration of diastase solution were added t o I O O cc. of 2 per cent soluble starch solution, incubated for 30 min. a t 40' C., then treated with I O cc. of 0 . 2 N NaOH. The soluble starch was prepared according t o Lintner, and its reaction so adjusted t h a t I O O cc. of t h e 2 per cent solution required o t o I cc. of 0.01 N NaOH t o make i t neutral with rosolic acid as a n indicator. By using t h e formula given b y Lintner (on t h e Lintner scale), allowing for t h e 'concentration of t h e enzyme, we can calculate t h e value of t h e d a t a obtained by the new method which would be comparable t o those obtained by thp original Lintner method. V : 0.1 = I O O : diastatic power (on Lintner's scale), since V = 0 , 2 6 2 , when t h e concentration of t h e enzyme is not j per cent (as used by Lintner) b u t Y per cent, we find t h a t : Diastatic power =
IO0
x
= Is4
0.1
0.262
Y
Y
If Y = I , then the diastatic value of t h e preparation used is 184 on the Lintner scale. The results of calculation t o t h e Lintner scale are shown i n Table I. TARLEI-SACCHAROGENIC POWER ovyzne (40' SCALE C FOR DIASTASE FROM AsPevgillzis
1
Lintner Obtained Scale X Y Value 735 3.5' 643 3.75 55 1 4.0' 505 4.25 459 4.5 413 4.75 367 5.01 336 5.25 305 5.5 275 5.751 262 6.0 249 6.25 236 6.5 223 6.75 210 7.0 197 7.25 184 7.5' 178 7.75 172 8.0 166 8.25 160 8.5 1.54 8.75 148 9.0 143 9.23 138 9.51 135 9.75 132 10.0 Experimental value.
Obtained Value 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 16.0'
17.0
18.0 19.0 20.0 22.0' 24.0 26.0 28.0' 30.0 33.0 36.01 37.0' 39.0 45.0 50.0 ,
Lintner Scale X Y 126 120 114 109 104 99 94 89 84 79 69 65 61 57 53 46 42 38 34 31 27 24 23 21 19 17
.
This table can be used for every form of diastase originated from A s p e r g i l l u s oryzae. If too much sugar is present in t h e diastase solution it is necessary t o correct the obtained Lintner value by a blank test. If the diastase is of different origin, the table may be changed somewhat since t h e course of digestion differs according t o t h e enzyme. The same procedure was followed t o develop a scale for t h e diastase of malt, using an extract of powdered malt, This scale is shown i n Table 11. Attention should be called here t o the fact t h a t t h e values falling between t h e experimental values were obtained by mere arithmetical calculations a n d not by mathematical interpolation. But, since this method is looked upon as a working factory method, t h e results
Oct., 1 9 2 0 TABJ.15
T H E J O 17 R N A L O F I AT D L'S T R I A L A N D ENGINEERIA'G C H E M I S T R Y
11-SACCHAROOENIC POWER SCALE FOR M A L T DIASTASE (40' C . ) Obtained Lintner Obtained Lintner Value Value X Y Value Value X Y 735 1 0 . 5 ' 3.51 92 55 1 3.75' 11.0 89 4 01 367 11.5 86 336 4.25 a3 12.0 305 12.5 4.5 80 275 13.0 4.751 77 262 5.0 75 13.5 249 14.0 5.25 73 236 71 14.5 5.5 223 15.01 69 5.75 210 16.0 65 6.0 197 17.0 61 6.25 184 18.0 6.51 57 177 19.0 53 6.75 170 20.0 49 7.0 46 163 21.0' 7.25 44 157 22.0' 7.5 151 40 24.0 7.75 36 144 26.0 8.0 138 27.0 8.25' 34 33 132 28.0 8.5 127 30.0 8.75 30 122 33.0 9.0 27 117 35.0' 23 9.25 112 36.0 22 9.5 107 39.0 20 9.75 102 45.0 18 10.0 16 50.0 Experimental value.
EXPERIMENTS A T 50' c.-In many cases, j o o C. is used as t h e temperature of t h e enzyme action, since t h e digestion of t h e starch is more rapid a n d there is less change i n slight differences in t h e temperature of digestion t h a n there is at 40' C.
TABLE VI-sACCHAROGEXlC
.
1
993
P O W E R SC.4LF: FOR THE DIASTASEO F
orqzae (SOo C.) Obtained Lin t n e r Obtained Value Scale X Y Value 3.251 952 12.75 13.0 3.5 833 3.751 7 14 13.25 13.5 4.0 595 476 13.75 4.251 4.5 14.0' 429 381 14.25 4.75 333 14.5 5.01 31 1 5.25 14.75 289 15.0 5.5 267 5.751 15.5 254 6.0 16.0 6.25 242 16.5 229 6.5 17.0 217 6.75 17.5 204 7.0 18.0 191 7.25' 18.5 178 7.5 19.0 I67 7.751 19.5 159 8.0 20.0 8.25 21.0 151 22.0 143 8.5' 138 8.75 23.0 133 9.0 24.0 128 9 25 25.0 123 9.5 25.5' 9.751 119 26.0 115 10.0 27.0 112 10.25 28.0 108 10.5 29.0 105 10.75 30.0 101 11.0 32.0 98 11.25 34.0 11.51 95 36.0 11.75 92 38.0' 89 40.0 12.0 86 45.0' 12.25 12.5 83 50.0' Experimental value.
Asgergiltus
Lintner Scale X Y 81 79 77 75 73 71 70 69 68 67 65 63 61 59 57 55 53 51 4948 45 42 401 37 3 5, 33 33 32 3 0' 29 28 26 24 21 19 17 14
are quite satisfactory. T h e best results are obtained b y the use of 6 t o 3 0 cc. digested starch solution (Lintner scale zoo t o 3 0 ) , which just reduce j cc. of Fehling's solution. Within these limits, t h e variation is rather small so t h a t even t h e mere arithmetical averages are entirely satisfactory. T h e following experiments will prove t h e a d a p t a bility of t h e new method for investigations of diastatic preparations. ( I ) TIME E F F E C T O N L I N T N E R vALuE-The influence of t i m e upon t h e Lintner scale values obtained by t h e new method was determined. T h e results shown in Table I11 indicate t h a t t h e Lintner value is practically proportional t o t h e digestion interval.
Table 1'1 has been worked out for j o o C. as t h e temperature of incubation.
TABLE I11
T.4sr.s VII-SACCHAROGENIC POWER SCALE FOR R.IAI,T DIASTAST;(50' C . )
Digestion Hrs. 0.25 0.5 0.75 1.o 1.5 2.0
Obtained Value
cc.
35.0 22.0 16.0 12.5 9.75 8.0
Lintner Scale 25 44 69 104 135 172
Time Factor 2 1 2/3
'12 1/3
l/a
1
Obtained Value 3.125' 3,251 3.51 4.0 4.51 5.0 5.25' 5.5 6.0 6.5 6.751 7.0 7.5 8.0' 8.5 9.0'
Lintner Scale X Time Factor 50 44 46 52 45 43
( 2 ) T I M E E F F E C T O N S U G A R CONCENTRATION-The sugar content of t h e samples was determined a t t h e end of I hr. T h e digested starch solution was kept for 48 hrs. protected with a cork stopper and t h e sugar content was determined again. (Further action of t h e enzyme, takadiastase in this case, was prevented b y t h e presence of t h e alkali.)
TABLE IV Samples
.
A............................. B............................. C............................. D............................ E............................
F.............................
--Obtained 1 Hr. 35.0 22.0 16.0 12.5 9.75 8.0
--
Valne 48 Hrs. 35.0 22.0 1h.O 17.5
9.75 8.0
( 3 ) R E L A T I O N O F TIME T O A M O U N T OF DIASTASE--AS shown i n Table V, t h e q u a n t i t y of diastase X period of digestion = constant. Takadiastase was used in t h e experiment. TABLE V Duration of Digestion Hrs. S t a n d a r d solution (10 c c . ) . . . . . . . . . . . . . 0 . 2 5 0.5 diluted solution ( I 0 cc.) . . . . . . . . . . . . 0 . 5 0.25 diluted solution ( 10 cc.) . . . . . . . . . . . . 1 . 0 0.125 diluted solution (10 cc.) . . . . . . . . . . . 2 . 0
Obtained Value 9.5 10.0
...
60.0'
... ... ...
,..
...
...
... 1
Lintner Scale X Y 666 500 333 283 233 199 182 173 157 141 133 129 123 117 108 100
11.01
12.0 17.0 14.01 15.0 16.0' 17.0 18.01 19.0 20.0 25.0' 30.0 35.0' 40.0 43.01 45.01 50.01
10
Lintner Scale X Y 96 92 83 77 71
6s. 62 58 54 501 4 8. 45 33 28 23 18 13 12 10
7
Experimental value.
c 0 sc I. uS I 0 s T h e method outlined is very convenient for experimental studies on various diastatic products, such a s malt a n d its various preparations, takadiastase, polyzime. etc. It is also convenient for t h e s t u d y of t h e influence of alkalinity, acidity, antiseptics, salts, etc., upon t h e activity of t h e diastatic preparation. T h e method has been used, as a routine factory procedure for over one year, with very good results. A C K N 0 W LE D G M E N T
Obtained Lintner Value Scale 18.0 61 18.5 59 18.5 59 19.0 57
I wish t o express my sincere t h a n k s to Dr. S. A. Waksman, of t h e Takamine Laboratory, for correcting t h e manuscript.
