EFFECT OF COLORING MATTERS
13.5'
nels is greater than the increase in the weight of the kernels, resulting in a higher percentage of nitrogen, also, in the outer kernels, but there were frequent exceptions to this. I t is apparent from these results that the kernels of the most desirable qualities came from the outer rows. Briefly summarized, the results of our studies of the composition of the grain from different parts of a single head show that the best grains from the standpoint of weight of kernel, and nitrogen content, came from the outer grains of the spikelets in the middle of the spike. Whether these properties are capable of transmission to the succeeding generations can be determined only by experimental trial, which we are inaugurating. LABORATORY OF
WASHINGTON
THE
AGRICULTURAL EXPERIMENT STATION, Pullman, Wash.
EFFECT
OF COLORING MATTERS ON SOME OF THE DIGESTIVE ENZYMES. B y H. W HOUGIITON Received M a y 29, 1907
T h e following investigation was conducted under the supervision of the Department of Chemistry of T h e George Washington University. I wish to express my indebtedness to Drs. C. E. Monroe and T. M . Price for their suggestions and assistance. A search through the literature indicates that there has been but little work done in the study of the effect of coloring matters on the digestive enzymes. However, that which has been accomplished by H . A. Weber', Edward Gudeinan3, A. J. Winogradow4, and others has given results which are exceedingly interesting and important. I n the article by H. A . Weber, it is shown that oroline yellow effects the peptic digestiou of fibrin, while saffoline, magenta and methyl orange effect the pancreatic digestion of this body. Gudenian found that ultramarine, burnt sienna, chrome yellow and ponceau effected the artificial peptic digestion of egg albumen when used in such quantities as one part, or less, of the color to 400 parts of the food. I n the investigation conducted by A. J . Winogradow, he found that Safranine, Ponceau R. R., Azofuchsine, G . , Orange 11, Coeruline S., Phoxine, R. 3. N., Iodeosine, Chrysaniline, Magdala red, Azoflavine, Presented before the meeting of t h e American Chemical Society a t Toronto, Ontario, June 27-29, 1907; being a thesia submitted t o The George Washington University for t h e Degree of Master of Science. On the Behavior of Coal-tar Color toward the Process of Digestion, by H. A. Weber, Am. Ch. J., 1596, XVIII, 1092-1096. Artificial Qigestion Experiments, by Edward Gudeman, J. Am. Chem. SOC. 190j.
The Influence of certain Coal-tar Dyes Z. Nahr. Genussrn., 1903,VI, $9-592.
011
the Digestion, by ,4. J. Winogradow.
I352
H. W. HOCGHTON
Benzopurpurine, aiid Cerise hindered the tiigehc,n of ;iibiinien by pepsiri even when only a few milligrams of thc color!; w?.creprcscnt ; an amount corresponding to from I : i o to I : I O O of the digestive solution. T h e action was almost inhibititive. T h e colors Quinoline yello\\-,Saphthylene green, Kaplithol yellon., Aniline Acid green, Iodine green, Acid azo-yellow T., green, Priniuline, Auramine o., Aniliiie orange. Martius yellow, and Metanil yellow interfered less than the first dyes, but in every case sonie effect was noticed. As stated, in these experiments the investigator used his coloring matter in proportion froin I : I O to I:IOO. This seems unpractical, as these colors are seldom used iu such strengths in practice. I t was only after an extensive review of the work of these investigators that the following investigation was undertaken in order to ascertain the effect of other coloring matters which have not as yet heen studied b\artificial digestion. 111 the investigations, the results of cvliich are recorded herein, t h e effects of annatto, saff roil, turmeric, cochineal, I3isinarcli brown and croceiiie scarlet I-I3 were studied on the peptic digestion of fibrin, casein and egg albumen. There was also studied the effect of ground annatto seetls and oil yellow on t h e action of the fat decomposing enzyme Iipa>e, lien i t was mixed with butter fat. T h e experi~nentswere carried out under laboratory conditions and it is not claimed that they represent exactiy t h e conditions which exist in a n animal, or that the phenomena n.ould occur iii precisely the same manlier in an anima! system, but it is heliel-ed that f r o m a consideration of these resillts n e 111ny iiifer, with a fair c!egree of accuracy, what woulci occur in an animal systelii, i f tlie bodies studied were introduced into it. The Effects of Coloring Matters on the Digestive Enzyme, Pepsin. I lie extent to wliici? t h e fihriii, cnsei:i. n r i t i ex9 nil)uii:en, used i n these experiments, were digested, was ascertained by deteriiiiilijig tlie a i i i o u i i t o f iiitrogen i n solution and t h e nrnount renrniniirg i n tlie residues :ifter tlic inaterials used I1nd stood ill contact with t!ie active fernleiits for two 1:rmr.q. The i:i trogeii in all sauip!e.i was deteri:ii:ieti !)y Gunning's Illetl:od, wiiich is one of tlie most accuxte methods known at the present r ,
d:1y.
PmjxzratioiL of Maisrials Used. TI?-. fibrin', was obtained by ivhipping freshly dranTn 1)Iood with n suitable instruiiicnt, the fibrin being dclmited R S a11 elastic, striugy nlnterial, a:hicll w n s freec.1 from adhering corpuscles b y tliorougli \vaslliiig aiid krleatlillg. Fibrin, so obtained, i y fici~iiip!i and iuiestinal inucosa. T h e storing and translocation of fats may be called ‘ lipogenesis,” the pro130th phases of lipogenesis cess being analagous to “ glycogenesis.” may be brought about by t h e enzyme, lipase, which is either fat forming or fat splitting, according to conditions. Pvejayatioz of M a t w i a l s Uscd-The lipase was prepared from calf liver in tlie following manner :’ Fifty grams of freshly chopped liver were weighed out and ground thoroughly in :i mortar with water and sand until well macerated. T h e mass was allowed to digest a t 40’ C. for one day. It was then filtered and niade up to roo cc. so a s to have a action,
Concernitig Lipaie, tlie fit-bl)littiny eiizvnie, aiid the revcrsillility of J. H. K:istle and A. S. Locvenliart, A1lll.Ch. J . , 24, 4 9 ~ - j 2 . j , igoo.
its
I357
E F F E C T OF C O L O R I N G R I A T T E K S
50 per cent. solution. A little chloroform and sodium carbonate were added to the solution so as to preserve its activity. T h e butter fat was prepared from double cream by extracting the fat with anhydrous ether to which a little sodium carbonate had been added. Mode of Procedzwc.--The first efforts were directed toward ascertaining the proportions in which each of the two coloring matters, annatto seed and oil yellow, were used for coloring butter. T h i s was arrived at by mixing the coloring matters with lard in various proportions. With annatto seeds it was found that a good butter color can be obtained between the proportions of 1:500 to I:IOOO. With oil yellow i t was found that agood color can be obtained between I:IOOO to I : I O ~ O O . These proportions are not precisely used in practice for coloring butter, as this fat has very often a natural color, but if these colors when used in the given proportions do not affect the hydrolysis of the butter fat, they will not when used in smaller quantities. I n these experiments the intermediate propqrtions between these ratios were used. I n each case one gram of the neutral butter fat was weighed out and the varying amounts of the coloring matters were added, as may be seen in the tables. BUTTERFAT A K D ANXATTO SEEDS. Proportions used ..................... o 1:500 1:1000 Amount of butter fat used ( g r a m s ) . .. I I I Amount of annatto used (grams). .... 0.0 0.002 0.001 Amount of lipase solution used (cc. ) 2 2 2 Amount of water used (cc.) .......... IO IO IO Length of digestion ( h o u r s ) . ........ 7 7 7 0 Amount of 1\'/20 HC1 used ( c c . ) . .... o 0 Amouiit of N/zo NaOH used (cc.) . 0.4 0.5 1.5 BUTTER FATAND OIL YELLOW. Proportions used.. ................ o I:IOOO 1:5000 1:2000 Amount of butter fat useti (grams) I I I I Amount of oil yellow used (grams) 0.0 0.001 0.0005 0.0002 Amount of lipase solution used (cc. ) 2 z 2 2 Amount of water used ( c c . ) ...... I O To IO IO Length of digestion ( h o u r s ) . ..... 6 6 6 6 Amount of N/20 HC1 used (cc. ) . o 0 0 0 Amount of 3 / 2 0 S a O H used (cc.) 0.5 1.8 1.2 1.0
..
.
1:2000 I 0.0005 2 IO
7 0
I
.6 I:IOOOO I 0.0001 2
IO
6 0
I .o
Conclusions. From the results shown i n the tables it may be seen that there weie no retarding effects caused by the coloring matters, annatto or oil yellow. As a matter of fact, each one aided the hydrolysis of the butter fat. There is no explanation to give for this increase of hydrolysis of the butter fat except that it might be suggested that the coloring matters contain some free acid. But this is not justified, as checks were made in each case. T h e only reason that can be given is that the coioring matters contain some lipolytically active substance as a part of their composition.' On the Action of Lipase, by Alonzo E. Taylor, J. Biol. Chem., 8 , 87-104,
1906.
