h I a y , 1914
T H E J O U R N A L OF 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
seems neutral t c litmus paper, a n d if this acidity be carefully neutralized, t h e diastase requires I j seconds more t o hydrolyze t h e starch a n d if now t h e starch be made t o have a n alkalinity equivalent t o 0 . 0 3 I per cent K O H , t h e activity of t h e pancreatin changes from I : 2 5 in 4 mins. t o I : 2 j in 7 l / 2 mins.. although t h e starch still meets t h e C . S. P. requirements a n d is neutral t o litmus paper. It would therefore appear t o one unaccustomed t o working with these enzymes t h a t t h e pancreatin had lost some of its diastasic activity, when t h e real t r u t h of t h e m a t t e r is t h a t t h e presence of t h e slight alkalinity in t h e starch inhibited t h e starch hydrolyzing power of t h e diastase. T h e percentage moisture in t h e corn starch on t h e market varies, b u t a difference of 2 per cent more or less is not a n i m p o r t a n t factor, as t h e following figures show. If t h e diastase tests I : 25 in j minutes on starch of 4.66 per cent moisture, it tests I : 2 j . j in j minutes on starch of 6.66 per cent moisture, or I : z j in 4 minutes 54 seconds: i. e., 2 per cent more moisture in t h e starch. or what is t h e same thing, 2 per cent less starch, showed t h e disatase t o have an acceleration of b u t 6 seconds. However, a difference in moisture content as great as I O per cent would show a decided effect upon t h e final strength of t h e diastase, b u t t h e average samples of starch would not v a r y t o such an extent. It is m y custom, before adopting a sample of starch for diastasic assay, t o wash it thoroughly with IO times its volume of distilled water a n d after drying t o determine its moisture a n d ash content, t h e n t o shake it u p with neutral, recently boiled a n d cooled, distilled water, a n d after filtering t o test t h e filtrate with cochineal indicator, a n d t o make use of t h e starch only when i t shows neutral t o this indicator. As a further check, I test a known sample of pancreatin with i t . A t h i r d precaution t o eliminate variations in assay, is t o use always a diluted iodine solution of t h e same temperature. A warm iodine solution does not show t h e starch iodide reaction so readily as a cold one. These three precautions t h e n should always be observed if uniform results are t o be obtained: I-The starch should be neutral or a t most b u t very weakly acid. 11-It should be chosen of average moisture content with a preference toward t h e use of anhydrous starch. 111-The temperature of t h e diluted iodine solution should be constant. DIGESTIVE FERMENTS COMPASY DETROIT
LABORATORY STUDIES ON M A L T EXTRACT' By HOWARD T. GRABER Some years ago I s t a r t e d a n investigation upon t h e concentrated glycerine extracts of malt t o determine t h e nature of t h e changes which t a k e place in malt extract upon aging a n d t h e causes of said changes. I was not successful in assigning a n y specific composition change a n d therefore did n o t submit m y results Presented at the 47th meeting of t h e A. C.
25-28, 1913.
S., Milwaukee, March
403
for publication. However, as there may be some points of interest in t h e work done, t h e results are puhlished herewith. For many years i t has been known t h a t malt extracts, of t h e concentrated glycerin variety, assume a dark color very soon after manufacture, and upon aging a year or more acquire a n acid t a s t e a n d a n odor of decomposition. The questions have been: ( I ) Does this change affect t h e diastasic power of t h e extract? ( 2 ) I s this change brought about b y a n y characteristic change in t h e composition of t h e extract? T o answer t h e first question, samples of malt ext r a c t , t h e diastasic power of which had been determined a t t h e d a t e of manufacture, were carefully selected from a series extending back twelve months a n d their starch hydrolyzing power again determined. Five grams of t h e extract were diluted t o I O O cc. with distilled mater a n d thoroughly mixed: I O cc. of this dilution were t h e n added t o I O O cc. of a z per cent starch paste of 40' C. a n d t h e mixture maintained a t 40' C. until hydrolysis was complete, noting t h e time required t o completely digest t h e starch; t h e point of complete hydrolysis being t a k e n a t t h e t time when one drop of t h e digested starch liquor failed t o produce t h e slightest trace of color when added t o 50 cc. of a solution containing 0.0006 g. of iodine a n d 0 . 0 0 1 2 g. of potassium iodide. T h e results follow:
7 9 7 10 10 14 7 8 9
7 15 13 10
10 14
7 8 9
12 months 12 months 10 months, 10 months 10 months 9 months 7 months, 7 months, 7 months,
15 days
20 days 13 days 5 days
9 9 7 7 8 8 8 9 7
9 9 7 7 8 8 8 8
7
months, months, months, months, months months, 5 months, 4 months 3 months 6 6 6 6 6 5
21 days 14 days 7 days 3 dags 20 days 14 days
All of t h e above extracts were very dark in color a n d most of t h e m h a d developed a disagreeable odor. It is evident t h a t t h e change which had taken place h a d not affected t h e diastasic, a n d therefore. t h e medicinal properties of t h e extracts. T h e second question is not such a simple one. T h e extractive m a t t e r dissolved from malted barley b y means of water and glycerin, consists of albumenoids, phosphates, maltose a n d dextrose, dextrin, a n d a peculiar principle termed diastase, as well as asparagine a n d carbohydrates of a higher rotary power called " malto-dextrine." T o form any conclusions as t o t h e n a t u r e of t h e changes taking place, most, if not all, of these ingredients should be determined. For this work I selected samples ranging in age from several weeks t o three years. T h e theory of t h e method used was: ( I ) T o exhaust t h e malt extract with absolute alcohol. From this alcoholic extract t h e percentage of maltose a n d dextrose as well as other d a t a were obtained. ( 2 ) T h e
T H E J O U R N A L OF 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
404
residue 'from t h e alcoholic extract after careful drying was exhausted with distilled water a n d from this water extract t h e percentage of. dextrins a n d albumenoids was obtained. T h e a p p a r a t u s used consisted of t h e usual Soxhlet extractor, with bulb condenser, cellulose filter about 4 inches long, sand which h a d been previously washed with hydrochloric acid a n d subsequently freed of acid, a n d absolute alcohol which h a d stood over unslaked lime while boiling 2 4 hours in a reflux condenser and distilled immediately before use. I n t o t h e cellulose filter introduce about 30 grams of t h e dried sand: filter a n d sand were carefully t a r e d upon a n analytical balance a n d upon t h e sand I O grams of t h e malt extract were accurately weighed. T h e filter with its contents was t h e n placed in t h e extractor a n d this was i n ' t u r n connected a t i t s upper end with a bulb condenser a n d a t its lower end with a 16 ounce distilling flask b y tightly fitting rubber stoppers covered with tinfoil. T h e 16 ounce distilling flask contained about I O ounces of recently redistilled absolute alcohol, a n d was shielded from t h e direct flame of a Bunsen burner b y a sand b a t h . T h e extraction of t h e malt extract b y t h e absolute alcohol was continued for about 48 hours a t intermittent intervals of 8 hours a t a time, or until a portion of t h e alcohol siphoning from t h e malt extract failed t o reduce Fehling's solution, showing t h a t all t h e sugars h a d been extracted, a n d also left n o weighable residue upon evaporation.
Vol. 6, No. 5
means of DeFrens' table given in Leach's "Food a n d Drug Inspection," pages j 9 j t o 597. This finished t h e work on t h e alcoholic extract. The apparatus consisting of t h e cellulose filter, sand, balance of t h e malt extract, together with t h e distilling flask were carefully dried; and, after drying were connected u p as before, using t h e same quantity of distilled water as of absolute alcohol in t h e previous extraction. T h e distillation was continued for another 48 hours a t 8-hour intervals or until a portion of t h e water siphoning from t h e filter did not leave a weighable residue upon evaporation. This constituted t h e second or aqueous extract. It was made u p t o 5 0 0 cc., thoroughly mixed a n d f r o m i t t h e following determinations were made: I-Total solids a n d 2-The percentage of dextrin a n d albumenoids. The total solids were obtained as in t h e alcoholic extract. The percentage of dextrins a n d albumenoids was calculated as t h e difference between t h e total solids a n d t h e per cent of ash. T h e other determinations made were moisture, ;acidity a n d diastasic strength. The acidity was determined by diluting 2 grams of t h e original extract t o 2 5 0 cc. with distilled water a n d titrating with N / I O sodium hydroxide using phenolphthalein as indicator direct, or if t h e solution was t o o dark t h e dropping plate was used, a n d t h e acid calculated t o lactic acid. T h e results follow.
(n
w
I
* 0
ALCOIIOLIC EXTRACT
L. u
AQUEOUS EXTRACT
:: E 2
s Y
."
-< 32 30 24 23 17 8 1
1.82 0.57 1.84 0.65 1.16 0.71 0.65
60.51 53.66 55.68 52.53 57.50 55.82 59.96
58.85 52.01 50.27 49.64 53.36 53.54 52.62
9.3 13.3 8.86 15.34 12.37 6.25 11.11
8.42 12.i 7.19 l3,50 10.8i 4.52 9.06
29 26 31.04 32.34 33.37 29.16 36.00 30.47
This alcoholic extract contains all of t h e sugars, some coloring matters with resins from t h e hops a n d some glycerin. It was evaporated t o dryness a t 80' C . . t h e residue dissolved in distilled water a n d t h e solution made u p t o j o o cc. with distilled water a n d filtered upon counterpoised filter papers. F r o m this solution t h e following determinations were made: r-The percentage of extract soluble in alcohol b u t insoluble in water. This was determined by drying a n d weighing t h e contents of t h e filter paper above. 2-The percentage of total solids in t h e portion soluble i n alcohol a n d soluble in water. This was determined by weighing 20 cc. of t h e solution in a t a r e d dish, evaporating t o dryness a n d weighing again. j-The percentage of reducing sugars was determined gravimetrically b y means of Fehling's solution, calculating t h e weight of reduced C u O t o maltose b y
0.88 2.04 1.6i 1.84 1,s 1,i3 2.05
101.7; 100.61
100 39 103.i3 101.69 100.51 104.24
99.23 98.36 93.25 99.00 96.05 96.50 94.85
0.ii 1.64 6.75 1.00 3.95 3.50 5.15
10 10 6 6 8 9 6
45 45 9 10 13 9 6
4 5 4.26
..
.. ..
0.09
CONCLUSIOh-S
I-Malt extract even after having changed t o a n almost black color a n d having a disagreeable taste is not necessarily inactive, a n d when properly made i t is potent for a t least one year. 11-Although t h e analysis of t h e extracts of differe n t age did not show a n y definite series of changes in sugar or dextrin content, no definite conclusions could be drawn as t o just what t h e nature of change was which took place. T h e results do indicate what t h e cause might be a n d further experiments have convinced me t h a t t h e cause of t h e deterioration of t h e diastasic power of malt extract is due t o t h e development of lactic acid a n d when t h e a m o u n t reaches a strength of I per cent or more i t greatly impedes starch hydrolysis. This developed acidity also undoubtedly causes t h e above mentioned blackening of t h e extract. DIGESTIVE FERMEXTS COXPANY DETROIT
