Oct.. 191 j
T H E JOCRNAL OF INDUSTRIAL A N D ENGINEERIXG CHEMISTRY CONCLUSIO~S
I-No common substance was found which could
853
are allowed t o ripen. Harz' found t h a t when t h e beans d o not ripen thoroughly or when t h e y are allowed t o ripen after t h e vines are c u t , starch m a y be present, certain varieties being more likely t o contain it t h a n others, whereas if t h e beans are thoroughly ripened t h e y are practically free from starch. Numerous authorities, including Schulze, Maxwell a n d Levallois, have identified galactan i n t h e soy bean, Levallois2 finding g t o 11 per cent. Likewise. Borghesani3 found from 2 . 8 6 t o 3.86 per cent pentosan in five varieties. For t h e past two years various varieties of soy bean have been grown on t h e Experiment Station F a r m . Calculated t o a uniform moisture content of I O per cent these showed compositions given in Table I. Typical analyses of commercial soy bean flours made in this laboratory also appear in Table I.
be used as a good substitute for lye in hulling corn. :----The analyses of several common brands of commercial lyc: are given. s---In practical tcrms it vias found t h a t not more t h a n z lbs. of lye t o 1 2 gal. of viater m-ere necessary t o good hulling a n d not less t h a n I lb. of lye t o 1 2 gal. of water could be used with good results. 4--Thcre need not be less t h a n a b o u t I bu. of corn t o 40 gal. of lye liquid a n d should not be more t h a n I b u . of corn t o 20 gal. of lye liquid. j.-- 7 0 0 C. ( 1 5 6 " F.) has been found a convenient temperature for hulling in about 1 ~ / 2 hours at t h e concentration just gii-en, b u t above 90' C. (194' F.) hulling can be accomplished in about 3 / 4 t o of t h e t i m e specified for 70' C. 6--Care must be observed in washing if all of t h e TABLE1 Protein lye is t o be remored from t h e corn. T h e a m o u n t of VARIETY Water Ash NX625 Fiber SOY BEANS GROWNO N EXPERIMENT STATION FARM: washing necessary has been roughly indicated. I t o S a n . , . . . . . . . . . 10.00 4.90 39.29 ;-It h a s been pointed o u t t h a t t h e process of hull- I t o S a n . . , . . , , . , , . 10.00 4.76 3 9 . 9 6 3.91 3.85 Quebec.. . . . . . . . . . . 10.00 5.26 38.24 3.57 ing is partly chemical a n d partly mechanical a n d t h a t Quebec.. .......... 10.00 5.64 40.46 3.68 Kentucky. . . . . . . . . 10.00 5.54 36.55 4.37 for t h e best results in hulling, very efficient stirring is Medium yellow., , . 10.00 4.68 40.99 3.79 M a n h a t t a n , . . . . . . . 1 0 . 0 0 7.77 40.66 3.67 necessary. E b o n y . . . . . . . . . . . . 10.00 5.16 40.59 5.21 UNIVERSITY O F MISSOURI, COLU>!BIA
THE CARBOHYDRATES AND THE ENZYMES OF THE SOY BEAN B y J. P. STREET A N D E. M. BAILEY> Received March 5, 1919
I n recent years various soy bean preparations have come into quite extensive use as special foods for t h e diabetic. T h e soy bean is especially suited for such a dietary as i t is very rich in protein a n d fat, a n d furthermore contains only traces of starch. N o t withstanding this almost complete absence of starch, analyses of commercial flour made from t h e soy bean invariably show from 2 0 t o 2 j per cent of nitrogenfree extract. A number of investigators have studied this nitrogen-free extract from various points of view, b u t as far as t h e writers are aware no complete separation of t h e different carbohydrates existing in t h e soy bean has been published. T h e form of these carbohydrates is vital t o t h e claims of t h e soy bean as a valuable food for t h e diabetic, for starch is b y n o means t h e only carbohydrate objectionable t o those afflicted with diabetes. T h e purpose of t h e present s t u d y , therefore, was t o a t t e m p t a rather complete quantitatiXTe separation of these carbohydrates in t h e material in question. T h e literature of t h e S O Y bean is very voluminous a n d no a t t e m p t \>-ill be made t o re\-ien- i t here. T h e published analytical d a t a are indeed conflicting. One author found 9.3 per cent sucrose, others only 3 o r 4 per cent, or less. Likewise certain European investigators report from 3 t o 5 per cent starch, while Japanese chemists have not identified starch in t h e native soy bean. hIany analyses of our own confirm t h e Japanese standpoint. These discrepancies as t o starch content are probably due t o t h e ripeness of t h e beans, or rather t o t h e method in which t h e y Credit for t h e chemical work reported in this paper is entirely due to Dr. Bailey. 1
N-free extract
Fat
6.29 4.85 5.83 5.09 5.19 6.88 4.67 5.64 5.33 5.98 5.73
37.70 40.66 38.56 37.38 35.07 39.15 3 7 . 75 37.23 33.15 35.59 38.49
5.87 4.10 4.73 5.78 5.81 4.64 4.98 5.43 5.03 5.46 4.28
26.64 25.67 25.86 24.88 26.82 25.27 23.58 24.97 27.11 24.55 25.02 27.56 29.55 26.30 28.42 28.98 29.59 26.97 28.44
AVERAGE.. . . . . . . 10.00 5 . 5 4 COMMERCIAL SOY BEAN FLOURS: 4.4 4.9 4.2 4.2 5.0 3.0 4 .6 4.4 f.8 4.4 4.1 6.5 4.6 5.1
38.29
4.64
26.64
14.89
45.7 43.1 42.3 42.3 39.9 41.0 42.9
1.9 2.2 5.4 4.7 3.9 3.4 4.2
22.6 24.9 24.5 25.8 24.9 25.0 22.4
20.5 21.4 19.8 18.2 19.1 20.0 20.8
42.5
3.7
24.3
19.9
..........
Peking.. 10.00 Medium g r e e n . , , , . 10.00 O k u t e . . . . . . . . . . . . . 10.00 Wilson. . . . . . . . . . . . 1 0 . 0 0 Arlington. . . . . . . . . . 1 0 . 0 0 Swan. 10.00 Morse. . . . . . . . . . . . 10.00 Cloud.. . . . . . . . . . . . 10.00 hIikado., . . . . . . . . . 10.00 Wing's mongol.. . . . 10.00 Hollybrook. , . . , , , 10.00
............
.- -
AVERAGE ........
5.1
4.5
-
~
15.26 15.76 17.07 15.34 16.72 15.27 14.32 14.07 13.03 15.84 15.86 14.19 14.38 13 .,03 14.18 12.72 16.90 16.00 13.06
-
A comparison of t h e analyses of t h e soy beans a n d t h e commercial soy bean flour shows t h a t t h e latter contains much more protein a n d f a t a n d considerably less of all t h e other ingredients, doubtless due t o bolting a n d other purifying manufacturing processes. T h e beans selected for t h e present investigation were of t h e Hollybrook variety a n d grown on our own farm. T h e following is a summary of their analysis expressed in percentages: TABLEI1
Protein N-free extract X 6.25) Ether extract and fiber TOTAL 36.69 14.92 31.08 100.00 COXSTITUENTS INCLUDED I N NITROGEN-FREEEXTRACT Undetermined hemicelluloses Dextrin, , , , . , . . , , , , , . . , , . . , Waxes, color principles, tannins, etc. (by diff.) . . . . . . . . . . . . . . . . . 8.60
zag : ;4
W
TOTAL.. .....................
Cellulose..
. . . . . . . . . . . . 3,29
Galactan f r o m raffinose.,. . . . . . . .
3 1.32 0.24
__
Per cent N-free Extract a n d F i b e r . , 31 .OS ( a ) Including 0.24 per cent galactan from raffinose.
T h e scheme of analysis in brief was t o t r e a t t h e finely ground meal successively with boiling 9 5 per cent alcohol, cold water, malt extract, I per cent hydrochloric acid, a n d 1 . 2 5 per cent sodium hydroxide, a n d Z e i : . Allg. Oesterr. Apoth. Ver., 23 (1885), 40. Comfit. rend., 93 (1881). 281. a Staz. sper. agrar.. 40 (1907), 118-120. 1 2
~
8 54
T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y
t o determine t h e kind a n d amount of carbohydrate removed b y each of these solvents. ALCOHOL EXTRACT
Vol. 7 , No.
IO
qualitative tests were made which bear on t h e identity of t h e sugar or sugars in t h e alcoholic extract. OSAZOKE FoRMATIOx-The simple hexose sugars in a s t a t e of purity form osazones in less t h a n 2 0 minutes. Sucrose forms glucosazones in about 30 minutes, b u t , as Browne a n d others have noted, t h e presence of organic matter, such as other sugars, greatly affects t h e rate of t h e osazone formation. After h e a t ing t h e direct solution a b o u t one a n d three-quarters hours a considerable amount of glucosazone was formed, m. p . 201-203' C., rapid heating (this should be 204-20 j'). According t o Tollens, raffinose yields a n osazone after about two hours, its m. p . being 187189". T h e osazone observed by us is apparently due t o sucrose, its formation being retarded for t h e reason noted above. I N V E R T I N TEST-The enzyme invertin hydrolyzes sucrose into reducing sugars; i t also acts upon raffinose a n d its failure t o produce reducing sugars a t all in t h e direct solution would indicate t h e absence of either of these sugars. It was found, however, t h a t 0.1 gram of invertin acting upon 2 cc. of t h e direct solution for I j minutes a t 6 0 - 6 j ' C. caused a heavy precipitate of CuaO with Fehling's solution. EMULSIN TEST-Emulsin hydrolyzes raffinose b u t is without action on sucrose:l 0.1 gram of emulsin I O cc. of water, incubated I O cc. of direct solution a t 38-40' C. for about t w o hours, gave a very decided increase in reduction over a blank in which t h e action of t h e enzyme was inhibited by boiling. F r o m t h e above d a t a , therefore, it is concluded t h a t raffinose is present, a n d t h a t we are justified in using Creydt's formula for sucrose in t h e presence of raffinose, in estimating these sugars from t h e polarimetric readings : Sucrose = 0.5188 a - b = 3.31 per cent, 0.8452 Raffinose = a - sucrose = 1.13 per cent, I.8j2 or, a total of 4.44 per cent as against 4.27 per cent obtained b y copper reduction as noted above.
T h e bean meal was freed f r o m f a t with petroleum ether, a n d t h e dried residue t h e n boiled under a reflux condenser with neutral 95 per cent alcohol f o r 8 hours. By this t r e a t m e n t reducing sugars, dextrose, levulose, invert sugar a n d maltose, a s well a s sucrose a n d raffinose, are extracted. Raffinose is less soluble in alcohol t h a n t h e other sugars named, according t o Beilstein one gram being soluble in 1000 cc. of cold 90 per cent alcohol, freely soluble in 80 per cent alcohol a t 60' C., a n d slightly soluble in boiling absolute alcohol. T h e same authority states t h a t 0.9 p a r t of sucrose is soluble in I O O p a r t s of 90 per cent alcohol a t 14' C., a n d one p a r t in 2 j 0 p a r t s of cold 97 per cent alcohol. From this it is seen t h a t while raffinose is much less soluble in alcohol t h a n sucrose, it is somewhat soluble in boiling absolute alcohol a n d , therefore, would be more so in boiling 95 per cent alcohol. It is estimated t h a t in t h e portions digested with t h e boiling 95 per cent alcohol not over one gram of raffinose was present in a volume of from 400 t o 450 cc. of alcohol. It seems reasonable, therefore, t o assume t h a t practically all of this sugar was removed a t this stage, especially a s i t is known t h a t sucrose a n d raffinose are in most cases removed together, a n d t h a t , in spite of their difference in solubility, i t is extremely difficult t o separate t h e m . T h e alcohol extract was evaporated nearly t o dryness a n d t h e n t a k e n up with water, t h e solution t h u s obtained not being clear. One cc. of basic lead acet a t e was added, causing a considerable precipitate, a n d t h e excess of lead removed with anhydrous sodium sulfate. A clear straw-colored solution resulted, in aliquots of which polarizations a n d reductions of Fehling's solution were made before and after inversion, T h e direct reducing power of t h e solution was very slight, a n aliquot corresponding t o 2 0 grams of t h e original material yielding only 0.0319 gram of CunO, equal t o 0 . 0 7 per cent of invert sugar. After inverting j o cc. of t h e solution in t h e usual way, a n aliquot C O L D TTATER E X T R A C T corresponding t o j grams of t h e original substance T h e alcohol-extracted material was dried a n d yielded 0.4367 g r a m of CUZO, equal t o 4.27 per cent weighed: 13.024 grams of this residue were digested of sugar, calculated a s invert sugar a n d sucrose. T h e direct polarization of t h e solution, calcu- with 2 0 0 cc. of cold water for 24 hours, ,filtered a n d lated t o t h e basis of normal weight of original material t h e water-insoluble material washed with about 2 0 0 in 100 cc. a n d read in a 2 0 0 mm. t u b e , was +5.4' V. cc. of cold water. T h e water extract was partially C.. a little sodium carbonate a t 14-15' C. T h e invert readings for t h e same a m o u n t evaporated a t 60-;0° being added to neutralize t h e faint acidity, a n d made of material were practically zero, t h e average of t e n readings being + 0 . 0 2 ' V. Polarizations of a duplicate u p t o a volume of 2 0 0 cc. T h e solution was opalescent alcohol extract prepared as above gave a direct reading a n d required t h e usual t r e a t m e n t with basic lead acetate before polarization. of 5.3 O V., a n d a n invert reading of +0.06' V. T h e direct reducing power of t h e solution was very T h e f a c t t h a t t h e invert readings were not decidedly minus indicates t h e presence of some sugar slight, a n aliquot representing 1.628 grams giving no visible copper reduction. After hydrolysis for two 5.4' other t h a n sucrose. A sucrose solution reading V. direct, on hydrolysis should read -2.3'. There a n d one-half hours with t h e usual amount of hydrois present, therefore, some simple sugar, or a complex chloric acid (sp. gr. 1.125), a n aliquot representing one which hydrolyzes into dextro-rotatory sugars, 0.814 gram gave 0.1710 gram of CuzO, t h e precipitate which counterbalance t h e levulose rotation. This being of a yellowish red color. This is equivalent sugar cannot be dextrose or maltose because of t h e t o 5.73 per cent calculated as dextrin in t h e original 1 Neuberg, Blochem. Z e z f . , 3, 5 19. slight direct reducing power of t h e solution. Three
+
+
+
+
T H E J O C R N - A L O F I S D C S T R I A L A N D EATGIA$TEERIiVGC H E M I S T R Y
Oct., 191j
material. I t will be shown later t h a t much of this reduction was due t o pentosan a n d galactan. Direct polarization on t h e basis of normal weight in I O O cc., in a 2 0 0 m m . t u b e showed + I I . j ’ V. a t IO’ C . ; after t h e usual inversion for I O minutes t h e j.;’ V., a n d after hydrolysis for t w o a n d reading was one-half hours i t was +4.4O, both readings a t IO’ C. T h e solution though opalescent gave a negative test for soluble starch. After adding strong alcohol t o a n aliquot of t h e water extract a n d allowing t o s t a n d for several days, t h e white gelatinous precipit a t e was filtered off, t h e filtrate evaporated, a n d t h e latter after making u p with water t o t h e same basis as above polarized +1.4’ V.,showing t h a t t h e strong rotatory power was due t o substances precipitable b y alcohol, such as dextrin, vegetable gums a n d mucilages. A test for mucic acid in t h e direct solution gave at first negative results, b u t after standing considerably longer t h a n t h e prescribed time a white crystalline residue was obtained of t h e general appearance of mucic acid ( m . p. 214’ C., r . h.). This indicates t h e presence in t h e water extract of a galactose-yielding sugar, or complex, besides dextrin, a n d might be d u e t o a small amount of raffinose not entirely removed b y t h e boiling alcohol. b u t more probably t o water-soluble galactan in t h e form of, or derived from, t h e vegetable gums a n d mucilages referred t o above. (The TTery close agreement in t h e polariscopic readings of t h e t\To extracts prepared with g5 per cent alcohol, t h e second one being digested twice as long as t h e first, however, makes i t improbable t h a t a n y raffinose could h a r e been left in t h e residue from t h e alcohol extraction.)
+
31.4LT
E X T R A C T DIGESTIOh?
Four grams of t h e material extracted with ether a n d boiling alcohol were washed with cold water first b y decantation a n d t h e n on t h e filter until t h e washings amounted t o about 300 cc. a n d failed t o reduce Fehling’s solution on t r e a t m e n t with hydrochloric acid. This residue was digested with freshly prepared malt extract i n t h e usual manner for two ninety-minute periods. T h e solution was t h e n h y drolyzed a n d its reducing power determined, showing t h e equixralent of 0 . jo per cent of starch in t h e original material. CELL
WALL
CONSTITL-ESTS
A C I D EXTRACT-The residues from t h e starch determinations were t h o r oughly washed with water, alcohol a n d ether, a n d dried. A t this stage all t h e more readily soluble sugars a n d t h e starch have been removed. a n d there remain t h e cell wall constituents. aiz., t r u e cellulose a n d those complex substances closely related t o cellulose a n d collectively termed “hemicelluloses.” T h e hemicelluloses are less resistant t o dilute acids t h a n t r u e cellulose, and t h e y yield copper-reducing bodies of t h e hexose a n d pentose types. I t is here t h a t t h e t r u e pentosan a n d galactan are found. I n t h e soy bean Schulze’ has shown t h a t t h e hemicelluloses give rise t o t h e pentose sugar arabinose a n d t h e hexose galactose. O S E PER
CEXT
HYDROCHLORIC
T h e above residue from t h e malt digestion was accordingly boiled with I per cent hydrochloric acid for one hour under a reflux condenser-this acid having been found t o be veryeffectiveinhydrolyzing pent0sans.l T h e solution was then filtered, t h e residue washed with hot water, t h e filtrate neutralized, cooled a n d made u p t o a definite volume. An aliquot equivalent t o 0.4 g r a m of t h e fat-free a n d alcohol-extracted substance reduced 0.0801 gram of CulO, equivalent t o 0.034j gram of dextrose, or j.93 per cent in t h e original material. As s t a t e d above, this reduction is not due t o dextrose. b u t t o t h e combined action of arabinose a n d galactose. With t h e d a t a obtained u p t o this point, we cannot s a y in what proportion these sugars are present. Their reducing powers, according t o Browne’s experiments,? are glucose mlucose -__ = 1.032, a n d b- - 0.898. arabinose galactose Calculating t h e reducing power obtained above as due entirely t o arabinose x e obtain 5 . i j per cent, or as d u e entirely t o galactose, 6.60 per cent. It is seen t h a t expressing t h e results as “sugars due t o hydrolysis of hemicelluloses, as dextrose” gives a figure close t o t h e mean. I n order t o gain d a t a for t h e more definite expression of t h e copper-reducing substances obtained a t this stage of t h e analysis, determinations of gala c t a n a n d pentosan were made. GALACTAX-Another residue from malt digestion was oxidized with nitric acid as in t h e official method for total galactan. X yield of 0.1480 gram of mucic acid (m. p. 212-214’ C., r. h.) was obtained, equivalent t o 3.38 per cent of galactose, or 3 . 0 j per cent of galactan on t h e original basis. PESTOSAN--A third residue from t h e malt digestion was treated in t h e usual way for furfurol-yielding substances, t h e equivalent of 3.09 per cent of pentosan, or 3.37 per cent a r a b a n being found. T h u s i t appears t h a t galactan a n d pentosan are present in practically equal amounts and, therefore, t h e reduced copper can best be expressed on t h e basis of t h e mean specific reducing powers of galactose a n d arabinose. Calculating in this way we obtain 6.18 per cent of carbohydrate which is due t o a galactoa r a b a n complex. This percentage agrees closely with t h e s u m of t h e percentages of galactan a n d a r a b a n . as determined. 1 . 2 j P E R C E X T S O D I U M H Y D R O X I D E EXTRACT-The residue from t h e hydrochloric acid t r e a t m e n t was t h e n boiled for 30 minutes with 1 . 2 j per cent sodium hydroxide solution, t h e filtrate being neutralized a n d hydrolyzed for two a n d one-half hours with hydrochloric acid. This filtrate was shown t o have no copper reducing power. C R U D E CELLULOSE-The residue from t h e previous t r e a t m e n t was thoroughly washed with water, alcohol a n d ether, a n d dried. It weighed 0.206j gram, equivalent t o 3.53 per cent on t h e original basis (not corrected for ash). T R U E CELLULoSE-This residue was t h e n moistened 1
1
Jour. P h y s . C k e m . , 1 4 (1890), 253.
85 j
2
Councler, Chem Z t g , 18 (1894). 1617 J . A m . Chem. S o c . , 28 (1906), 439.
856
T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y
with water a n d exposed t o chlorine gas for one hour, t h e n washed with water, boiled in a solution of 2 per cent sodium sulfite a n d 0 . 2 per cent sodium hydroxide, filtered, washed a n d dried. T h e residue from this t r e a t m e n t weighed 0.1945 gram, equivalent t o 3.34 per cent on t h e original basis (not corrected for ash). The ash of this residue weighed 0 . 0 0 2 7 gram, or 0.05 per cent on t h e original basis, making t h e t r u e cellulose 3.29 per cent. D I S T R I B U T I O N O F P E N T O S A N S A N D GALACTANS
Distinct from t h e insoluble hemicelluloses of t h e cell wall constituents are t h e soluble pentose- a n d hexose-yielding substances. I n this group, broadly termed saccharo-colloids b y Tollens, belong t h e vegetable gums a n d mucilages, which, from t h e nature of their transformation products, must be regarded as pentosans a n d galactans. They are soluble in water a n d generally insoluble 111 strong alcohol, though some appear t o be dissolved t o a slight degree in t h e latter It should not solvent, especially if somewhat dilute be forgotten, of course, t h a t certain t r u e sugars yield mucic acid a n d furfurol due t o some particular complex in t h e sugar molecule. T h u s raffinose yields mucic acid a n d sucrose yields furfurol, due t o t h e galactose a n d levulose complexes respectively. According t o Schulze, water-soluble pentosans are present in small amount in t h e soy bean. We have endeavored t o gain some idea of t h e distribution of pentosan a n d galactan b y determining t h e mucic acid a n d furfurol yields in t h e hot-alcohol-extracted a n d in t h e water-extracted a n d malt-digested residues. I n respect t o mucic-acid-yielding substances t h e t o t a l galactan found was 4.86 per cent. I n t h e alcoholextracted material 4.62 per cent was present a n d in t h e water-extracted a n d malt-digested residue 3.0 j per cent. It appears, therefore, t h a t a small amount of t h e mucic-acid-yielding substance was removed b y hot 95 per cent alcohol. This is attributable t o t h e galactose complex in t h e raffinose molecule. Extraction with water a n d digestion with malt removes 1 . j 7 per cent, showing t h a t galactose arising from water-soluble galactan was in part responsible for t h e copper-reducing power of t h e hydrolyzed water ext r a c t already noted. There remains 3.05 per cent, or 62.7 per cent of t h e total galactans, in t h e cell wall , constituents. With regard t o pentosans there were found in t h e original soy bean meal furfurol-yielding substances equivalent t o 4.94 per cent pentosan. H o t alcoholic extraction removed 0.83 per cent, a n d 1.02 per cent was removed b y t r e a t m e n t with water a n d b y malt digestion. T h u s 3.09 per cent, or 62.5 per cent of t h e t o t a l pentosan, remains in t h e cell wall constituents. T h e small amount removed by alcohol is largely due t o substances insoluble in water, precipitated or occluded in t h e lead precipitate, when t h e alcoholic extract is t a k e n up with water a n d treated with basic lead acetate. This lead precipitate was found t o yield copper-reducing matter equivalent t o 0 . 5 per cent pentose. T h e nature of t h e saccharo-colloids is rather obscure b u t t h e y are quite probably t h e vegetable gums a n d mucilaginous materials present in seeds
Vol. 7, No.
IO
a n d plant tissues, which are dissolved in a slight degree by alcohol, a n d which are removed more completely by water in t h e next stage of our analysis. T h e removal of fructose as such, or in complex, m a y also offer a partial explanation. T h e 1 . 0 2 per cent of pentosan removed by water contributed t o t h e copperreducing power of t h e hydrolyzed water extract. ORGANIC ACIDS
Organic acids are largely precipitated b y lead acetate, a n d t h e a t t e m p t was made t o determine these b y t h e scheme outlined by Schulze, viz., precipitating t h e aqueous solution of t h e alcohol extract with lead, removing t h e excess of lead, decomposing t h e lead precipitate with hydrogen sulfide, removing the excess of t h e latter, a n d t h e n precipitating t h e acids with calcium or strontium. An a t t e m p t t o precipitate these acids with calcium acetate, as is done in t h e case of malic acid, failed for some unknown reason. Strontium hydroxide, however, gave uniform results, b u t somewhat lower t h a n expected although there are no recorded analyses for comparison. T h e lead precipitate from j o grams of material was decomposed with hydrogen sulfide, a n d t h e excess of t h e l a t t e r removed from t h e filtrate by evaporation. T h e clear, white solution was made u p t o 5 0 0 cc. a n d aliquots of 50 cc. t a k e n for analysis. T h e aliquots were evaporated t o 2 0 cc., 2 t o 3 cc. of a saturated solution of strontium hydroxide a n d 180 cc. of g j p e r . cent alcohol were added, a n d t h e mixture was heated on t h e steam b a t h for one hour, t h e n filtered while still hot. T h e precipitate obtained was yellowish in color, t h e filtrate being clear a n d giving no further precipitation with additional strontium hydroxide. On igniting t h e precipitate a n d titrating t h e strontium oxide 1.44 a n d 1.29 per cent of organic acids, calculated as citric. were obtained. UNDETERMINED SUBSTANCES
T h e amount of nitrogen-free extract which remains unaccounted for is 8.60 per cent. This is attributed largely t o those undetermined substances removed b y alcohol, since there was obtained about 16 per cent of alcohol extractives of which a little more t h a n onethird have been determined. T h e exact n a t u r e of t h e undetermined extractives cannot be s t a t e d with much certainty, b u t t h e y undoubtedly include chromogenic substances, vegetable waxes a n d possibly t a n n i n complexes. It has already been noted t h a t basic lead acetate produced a voluminous precipitation of these alcohol-soluble materials, b u t the purpose of this investigation has been served in demonstrating t o what extent t h e y were copper-reducing or yielded copper-reducing bodies on hydrolysis. It has been shown t h a t t h e amount of such substances was small. We shall include these undetermined extractives in t h e alcohol fraction through lack of d a t a for their distribution. S U 41MARY
A consideration of t h e above d a t a shows t h a t t h e meal examined contained 4.51 per cent total sugars, 0 . j O starch, 3.14 dextrin, 4.94 pentosan, 4.86 galactan (less 0 . 2 4 due t o raffinose), 3.29 cellulose, 1.44 organic
Oct., 1915
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 EiVGIiVEERIA\TG C H E M I S T R Y
857
direct suspensions of t h e meal in water were allowed t o a c t upon a X / z solution of sucrose for periods of time u p t o 18 hours a t temperatures ranging from 20° t o jj". PROTEASE--NO gelatin-liquefying action was detected b u t in trials with Witte peptone a t 40' and with H C N as t h e antiseptic positive t r y p t o p h a n e tests were obtained after 48 hours, t h e same being very marked after 7 2 hours. Trials in which t h e peptone was omitted gave negative tryptophane tests, indiTABLE III-SUMMARY O F . h A L Y S I S O F N I T R O G E K - F R E EXTRACT E A K D FIBER cating no action upon t h e native proteins of t h e soy HOT 95 P E R CENTALCOHOL EXTRACT: Insoluble in water or precipitated by lead acetate: bean itself. Saccharo-colloids a s pentosan . . . . . . . . . . . . 0.83 . . . . . . . . . . . . . . . 1.44 Organic acids, as c i t r i c . . . . . . . OXIDASES-~f'ater suspensions of t h e meal decomWaxes, color principles, etc., b y difference.. . . . . . . . . . . . . . . . . .8 . 6 0 posed hydrogen peroxide slowly at room temperature. Soluble in water a n d not precipitated by lead acetate: Invert sugar . . . . . . . . . . . . . . . 0.07 T h e y were without effect upon tincture of guaiacum Sucrose ( C r e y d t ) , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.31 Raffinose (Creydt) (0.24 galactan derived from galactose comexcept in t h e presence of hydrogen peroxide, when a plex in raffinose). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.13 blue coloration was rapidly produced. COLD WATERE X T R A C T : Soluble s t a r c h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 .OO LIPASE-There is a distinction between those enzymes Reducing matter after hydrolysis, calculated as dextrin (including 1.57 galactan a n d 1.02 pentosan). . . . . . . . . . . . . . . . . . . . . . . . . . 5.73 capable of decomposing various esters ibutyrases) MALT EXTRACT: Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.50 a n d t h e t r u e lipases which act upon t h e natural fats. 1 PER CENT HYDROCHLORIC ACID EXTRACT: Hemicelluloses, reducing matter calculated as galacto-arabinose, T h e soy bean was found t o effect both of these reac(Determined directly in the malt residue, galactan 3.0.5, pentosan tions. . T r u e lipases have been found in t h e seeds of as araban 3.37). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.18 1.25 PER CENT SODIUZll HYDROXIDE EXTRACT: Ricinus, rape, opium poppy, h e m p , flax a n d maize, Reducing sugars.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.00 CHLORINATED RESIDUE,TRUEC E L L U L O S E. . .. . . . . . . . . . . . . . . 3 . 2 9 a n d are presumably present in all oily seeds. T o r . 4 ~. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.08 T h e first trials made were with t h e turbid filtrate prepared as described above. T h e substrate used T H E E S Z Y M E S O F T H E SOY B E A S was butyrin ( AIerck's) a n d t h e antiseptic, toluol. A n u m b e r of investigators have reported t h e presPortions equivalent t o one gram of material were ence in t h e soy bean of r a r i o u s enzymes. T h e most allowed t o act on 0 . 2 5 cc. of butyrin a t i o o , all trials conspicuous a n d i m p o r t a n t is t h e urease of Takeuchi,' being neutralized t o phenolphthalein a t t h e beginning, which acts specifically upon urea, liberating ammonia. a n d t h e acidity subsequently developed determined He has called a t t e n t i o n t o t h e economic possibilities by titration w i t h s,Io sodium hydroxide ~~taq-hour of this enzyme from a n agricultural standpoint, a n d intervals. T h e duration of t h e esperinient w a s Van Slyke a n d others? have made practical analytical 96 hours. T h e results can best be expressed b y a use of i t in t h e form of standardized urease prcparatabulation of some typical trials a n d their controls tions for determining nitrogen in urea-containing sub(Table IV). stances, such as blood, urine a n d other animal secreT A B L E IV-THE ACTIO&' O F SOY BE.4N I'REF.4R.%TIOS I V I T I i l i U T Y K I N tions a n d juices. Sting1 a n d hlorowski3 have reported SOLUTIONS TESTED Cc. Y/10 N a O H required after t h e presence of a powerful amylase. Bertrand a n d Toluol added to all 24 48 72 96 h-0. Butyrin IVater Addition Hrs. Hrs.. Ilrs. Hrs. Total Riokind' discovered a glucoside, vicianin, a n d a n I 0 25 cc. 2 0 cc. 0 1 0 I 0 I 0 . 1 0l: 2 Soiie Xone 20 cc. extract O.,i 1 . 1 0 . 8 0 . 7 2.') enzyme capable of effecting its hydrolysis. 3 0.2.5 cc. S o n e 20 cc. extract 2 . i 1.5 1 . 2 I . ? 6 . 4 1. I 1 . 0 1 . 0 1.0 4 . 1 I n this examination of soy bean meal a number of 4 0 . 2 5 cc. None 20 cc. boiled extract 5 0 . 2 5 cc. 20 cc. 1 gram meal 5.5 2.5 . , ,3 0 1 1 . O additional enzymes have been found. T h e y will Sa hTone 20 cc. 1 gram meal 1.J 1.0 . . 4.4 6.9 6 None 20 cc. 0.5 gram glycinine 0 . 3 0 . 3 0..3 . . . . 0.5, be mentioned only very briefly since t h e wide distribution of (enzymes in seeds a n d other vegetable I t is apparent t h a t following t h e course of lipolysis tissues a n d t h e readiness with which t h e y c a n , in most b y means of pericdic titrations with alkali is comcases, be detected, make a survey of such materials plicated b y t h e acid production of t h e soy bean, for t h e detecrion of enzymes of decreasing interest. in substance, a n d its extracts both boiled a n d unboiled. T h e meal was ground with s a n d a n d distilled water, While t h e acidity developed in Trials 3 a n d ;. was t h e suspension filtered through cheesecloth a n d t h e greater t h a n in t h e various controls, t h e very cont u r b i d filtrate used in most of t h e trials recorded. siderable a n d jrregular acid development in t h e latt,er AMYLASE-^^^ aliquot representing one g r a m of is a disturbing feature. T h e development of acidity material completely transformed 0 . I g r a m of soluble b y cereal grains is generally recognized a n d one of u s starch in 3 hours a t 5 j O , while with 0.I g r a m of material ( J . P. S.) has given some attention t o this point in acting on a n equal weight of substance t h e t r a n s - a s t u d y of t h e determination of acidity in cattle formation reached t h e erythrodextrin stage, as indi- feeds,' b u t t h e satisfactory explanation of t h e phenomcated b y iodine tests. enon is not yet presented. T h a t t h e protein of t h e SUCROSE-POlariSCOpiC readings indicated no hydro- soy bean is not t h e disturbing factor is shown b y Trial lyzing action upon cane sugar. Turbid filtrates a n d 6. T h e most pronounced ester decomposition is exI Jour. coll. A s Y . ,L'nia. T o k y o , 1 (1909), 1. hibited during t h e first z4-hour interval. some re2 Jour. B i d . Chem., 19 (1914). 2. tarding influence being exerted thereafter. .IlonuLsrh. f. Chem., April, 1886. acids a n d 8.60 per cent waxes, color principles, etc. Of these constituents only t h e first three, z ~ i z . t, h e sugars, starch a n d dextrin, amounting t o 8.1 j per cent, m a y be considered objectionable in a strict diabetic diet. T h e remaining nitrogen-free extract, 22.93 per cent, a b o u t three-fourths of t h e whole, consists of pentosans, galactans, organic acids, waxes, etc., forms which are n o t generally considered a source of danger i n a diabetic diet.
~
B i ~ i i .Sci Phorm., 14 (190;),
161.
: U.
S.Dept. 4gr.. Bur. of Chem., Buil. 122 (1909), 160.
T H E J O U R N A L O F I N D U S T R I A L A h - D ELYGINEERING C H E M I S T R Y Another experiment was tried following t h e suggestion of Reynolds Green (“Soluble Ferments,” p. 2 2 7 ) . Five grams of fat-free material were extracted with IOO cc. of 5 per cent NaCl containing 0 . 2 per cent K C S for -2 hours at 3j-40’. T h e preparation v a s filtered a n d trials made ( I ) directly with t h e filtrate a n d ( - 2 ) with t h e filtrate after “actirating” b y making slightly acid with dilute acetic acid a n d allowing t o T h e unactivated s t a n d z hours longer a t 3j-40’. filtrate ( I ) showed no pon-er t o hydrolyze b u t y r i n ; t h e activated filtrate ( 2 ) was decidedly active. T w e n t y cc., equivalent t o one gram of soy bean meal, acting upon c . 2 j cc. butyrin required a t 24-, 48- a n d j z - h o u r periods 2.0: 1.7 a n d 2 . 0 cc.: total 5.7 cc.: N/ION a O H t o neutralize t h e acidity developed. T h e control in 15-hich t h e extract h a d been boiled required a t corresponding inter\-als 0 . 6 , 0.7 a n d 1.0cc., or a total of 2 . 3 cc. This activated extract also showed lipolysis of nn emulsion prcpared v i t h soy bean oil. I t appears t h a t t h e spontaneous acid production of t h e soy bean itself h a s been largely eliminated b y t h e above process. T h e amounts of acid developed b y t h e control are uniform a n d much less t h a n in previous controls, a n d t h e decomposition of butyrin is uniform a n d progressive, not diminishing as in previous trials. There is present t h e n a t r u e lipase. which is dissolved b y j per cent XaC1 a n d n-hich is either activated b y acetic acid or generated b y t h a t acid from its zymogen. co~cLusIos I n addition t o t h e urease, amylase a n d glucosidesplitting enzyme reported by other workers, t h e soy bean contains also a protease of t h e peptoclastic t y p e , a peroxidase a n d a lipase. Negative results have been obtained for sucrase a n d protease of t h e peptonizing type. I t was thought unnecessary t o examine t h e material for urease a n d no a t t e m p t n-as made t o corroborate t h e presence of t h e glucoside-splitting enzyme. The presence of a n active amylase h a s been corroborated.
Val. 7 , h-0. I O
found t h a t t h e method is altogether t o o exacting in order t o be serviceable in technical work. Unless t h e determination is carried out with t h e utmost care t h e results fail t o be reliable. Owing t o t h e varying composition of t h e gelatine employed a n d t h e exacting conditions for performing t h e experiment, such as a fixed incubation temperature at n-hich t h e solution is t o be kept for a number of hours, t h e necessity of ice for chilling t h e water, t h e special preparation of a bacterial acid solution, etc., I have found t h a t , when employing this method i n practice, considerable difficulties are encountered. As a m a t t e r of fact i t seems almost impossible for two chemists working independently a n d each employing reagents of his own preparation, t o arriT-e at similar results. Although I have h a d occasion t o experiment with almost every one of t h e known methods for determining t h e extent. or progress, or products of proteolysis in malt solutions. such as t h e Fuld-Levison-Edestin method. t h e electrical conductivity method, e t c . , I have found t h a t , aside from t h e gelatine method above mentioned. there is only one other method which is applicable t o malt solutions. This method is t h e one I have employed a n d has for its basis t h e Soerensen formaldehyde titration as originally published in Biochem. Ztschr.. 7 (1908). 4 j . It has been so modified by me as t o greatly simplify manipulation in order t o p u t t h e method within t h e reach of every one accustomed t o relatively simple laboratory work. By t h u s simplifying t h e method with only a comparatively slight sacrifice as t o accuracy, I have been able t o reduce experimental error t o a mini m u m so t h a t t h e results obtained will be of such degree of correctness as t o be equal in every respect t o t h e usual standard of accuracy maintained in technical analysis. Furthermore, t h e preparation of t h e different reagents has been greatly simplified, a n d t h e entire work so arranged t h a t t h e estimation does not require more t h a n a total of j o minutes in time. A N A L Y T I C A L L.4BORATORY T h e principle upon Thich t h e formaldehyde method C O N X E C T I C U T t \ C R l C U L T U R A L E X P E R I M E N T STATIOX depends is t h e estimation of reacting carboxylic-acid h-i~rnHAVEN groups present in a digestion mixture. An amino . acid, owing t o its amphoteric character, cannot be ACID RATIO : A NEW METHOD FOR DETERMINING THE directly titrated by a s t a n d a r d alkali solution in t h e PROTEOLYTIC STRENGTH OF GERMINATED presence of a n indicator. If, however, formaldehyde GRAIN 1N TECHNICAL ANALYSIS in excess be added before t h e titration, t h e amino B y CARL A. SOWAK Received bray 10, 1915 groups are converted into methyleneimino groups, T h e proteolytic activity of malts has been studied losing their amphoteric character, whereupon t h e y can extensively b y R . W a h l , t h e results of whose experi- be directly t i t r a t e d with standard alkali in t h e presence ments were published in t h e P i o c e e d i i z g s of t h e 8t1~ of phenolphthalein. T h e result of this titration is usually expressed as I n i e r n a t i o ? z a l C o i i g y e s s of A p p l i e d C h e m i s t r y , 14, 21j--220. I n this paper D r . Wahl gives t h e t h e percentage a m o u n t of total nitrogen in a mixture details of a method devized by him, based upon t h e which enters into reaction with formaldehyde. For principle first made use of by Schidrowitz; namely, t h e purpose of malt valuation this is, however, not t h a t t h e proteolytic enzymes exert a liquefying action necessary, as we are not so much concerned with abupon semi-solid gelatine a n d t h a t t h e relative pro- solute values as with relative values. VV7hat we wish teolytic strength can be measured b y t h e extent of t o determine is which malt or t y p e of malt yields t h e this liquefying action. B y using this method Wahl greatest a m o u n t of amino acids, a n d also t h e extent has arrived a t a series of very valuable results regarding t o which this degradation of t h e proteids proceeds t h e peptic powers of various malts prepared from dif- within a given time in different types of malt. I t is, ferent t y p e s of barleys. T17hile I have made use of therefore, equally serviceable a n d far more convenient Wahl’s method on a number of occasions, I have t o make use of t h e titration figures a s t h e y are ob-
