1 - 0IT., I 9I3
T H E J O 1 7 R S d L O F I S D L - S T R I A L . 4 S D ESGIAVEERILVG C H E M I S T R Y COLOR
S U G G E S T I O S S AS T O T H E C.AL*SES F O R C O L O R C H - I S G E S
Ortho-tolidine is a n organic compound of t h e formula C14H11,S2.1I t is a homolog of benzidine a n d is a powder melting a t 1 2 9 O C. I t m a y be prepared from o-nitrotoluene b y reduction with zinc dust a n d sodium hydroxide. T h e resulting hydrazo-toluene is converted i n t o t h e o-tolidine b y boiling with hydrochloric acid. I t m a y be purified in a manner similar t o t h a t of benzidine. I t s structural formula m a y be written as a t ( I ) : 1
OF
S e u t r a l alcoholic soI u 1ion Aniline Yellow-green-brown h-H?
917
PRECIPIT.4TE IX Alcohol a n d acetic acid. Brown-purple-red
Alcohol a n d HC1. Yellow-purplr-red
0 o-Toluidine I'ellow-orange-brown
3"'^
Purple-blue-red
Purple-red
SH2
.,
Benzidine Yellow -oran gc-red SH?
0
I t is a p - 2-diamido-nz-2-dimethyldiphenylcomp o u n d , a n d probably produces dyes of t h e same general t y p e a s those derived from benzidine. Oxidation of o-tolidine probably produces a nitroso compound of formulb ( 2 ) : this compound is probably blue in color. Salts of o-tolidine upon oxidation produce a yellow dye. a possible formula for t h e acetic acid salt being ( 3 ) . T h e green color produced b y t h e action of t h e chlorine on t h e acetic acid solution of o-tolidine is probably a mixture of t h e blue compound ( 2 ) a n d t h e yellow compound 13). I n t h e hydrochloric acid solution of o-tolidine. HCl is merely substituted f o r H O O C C H , . T h e slight dissociation of acetic acid a n d t h e hydrolysis of t h e acetate perhaps account f o r t h e slow formation of t h e yellow dye in t h e acetic acid solution, while t h e large dissociation of hydrochloric acid might be t h e reason for t h e rapid development of t h e yellow color in t h e hydrochloric acid solution. T h e red color a n d red precipitate produced b y large a m o u n t s of chlorine m a y be a substitution product of t h e nitroso compound resulting from t h e complete oxidation of all t h e o-tolidine present. Xniline t r e a t e d with a solution of bleaching p o n d e r produces certain color reactions, b u t i t requires a considerable a m o u n t of chlorine for their development. Si mi 1ar 1y or t h o - t ol ui din e for m s c ol or e d solutions with chlorine. \T-hich are likewise slon-ly developed. although someivhat more rapidly t h a n in t h e case of aniline. -4s has been previously noted benzidine readily produces colored solutions n-ith chlorine, similar t o those formed with o-tolidine. I t s sensitiveness t o chlorine is much greater t h a n is t h a t of aniline or o-toluidine b u t somewhat less t h a n t h a t of o-tolidine. T h e colors produced b y a solution of bleaching powder in gradually increasing quantities with neutral a n d m-ith acid alcoholic solutions of aniline, o-toluidine, benzidine a n d o-tolidine are shown in t h e following table: 3rd edition, Vol. 4, page 980
1-ellow-orange-red
Green-orange-red
tellow orange-red
A
0
1 Beilstein,
Green-orange-red
SHz o-Tolidine Y e l l o w - o r a n p r e d
OCH3 SH:
X b!ue color reaction produced b y t h e action of hypochlorites o n methyl-aniline and ethyl-aniline h a s been recently reported b y Leech.' H e suggests t h a t t h e blue dye. possibly a member of t h e "indo" series of dyes. results from t h e oxidation of t h e methyl or ethyl group. It seems t o t h e authors t h a t in t h e case of o-toluidine a n d o-tolidine t h a t t h e dyes are produced as a result of t h e oxidation of t h e amido group, a n d n o t t h e methyl group, since in aniline a n d benzidine. having no methyl groups, similar dyes are formed b y t h e action of hypochlorites. CISCISSAT1 F I L S R A T I O h - P L A N T CINCINNASI, OHIO
THE QUANTITATIVE ESTIMATION OF GLIADIN IN FLOUR AND GLUTEN B y GEO. A. OLSON Received July 26, 1913
Since Einhof? separated alcohol-soluble proteins from wheat, r y e a n d barley, much interesting research work has been done on prolamines. It was n o t . however. until Osborne a n d Voorhees? presented a s t u d y on t h e proteins of t h e wheat kernel t h a t a clear understanding as t o t h e n a t u r e of vegetable proteins was established. Of particular interest was t h e research on gliadin. t h e prolamine of wheat. It has been further belie\-ed t h a t t h e proportion of gliadin t o glutenin determines t h e quality of t h e gluten which in t u r n 1 "A Color Reaction of Hypochlorites with Methyl-Aniline a n d EthJ-1. m . Chrm. Soc., 36, No. 8 (August, 1911). Aniline," b y Paul S . Leech, J O U YA 2 See "The Proteins of t h e Wheat Kernel a n d Vegetable Proteins," by T h o m a s B. Osborne. Also Trans. of the Canadian Instilufe, 7, 1903, b y George G. Nasmith. A m e r . Chem. J . , 16, 392 (1893).
91 8
T H E J O L7R N d L 0 F I N D r S T RI ;I L ili1-D E S G I N E E R I N G C H E M I S T R Y
determines t h e quality of a flour for bread-making purposes.’ Following Millon’s* work on t h e q u a n t i t y of gluten contained in different kinds of wheat, another series of investigations bearing on t h e relationship of t h e gluten t o t h e baking qualities of flour was commenced. Heinrichs3 failed t o establish a n y relationship between t h e gluten a n d baking quality, b u t Fleurentl was led t o believe t h a t t h e alcohol-soluble portion of t h e gluten, rather t h a n gluten, determines t h e baking qualities of flour. T h e method adopted b y Fleurent for t h e extraction of gliadin cannot be said t o be satisfactory since t h e diluted alcohol contains potassium hydroxide a n d potassium hydroxide like sodium hydroxide is a solvent for glutenin as well as for gliadin. Based upon Osborne’s a n d Voorhees’ work, Teller5 worked out a method which h e used for t h e estimation of gliadin in wheat a t different periods of growth. He, a n d subsequently Chamberlain6 a n d others,: observed t h a t a p a r t of t h e alcohol-soluble nitrogen was extracted b y salt solution. T h e writer i n his studies on t h e nitrogen components of wheat flour has confirmed this work. T h e fact t h a t t h e quantitat i r e methods which we follow for t h e estimation of proteins in wheat flour are far from satisfactory, led t h e writer t o carry out a series of experiments, some of t h e results of which are recorded below. T h e strength of alcohol most suitable for t h e extraction of gliadin from flour, or gluten, has never been determined. It is known t h a t above certain strengths of alcohol little or n o gliadin can be extracted, while below this limit all t h e gliadin can be extracted, provided enough solvent is used. Teller’ studied t h e solubility of proteids in different strengths of alcohol a n d found t h a t while t h e quantity of nitrogen extracted increased with t h e dilution of alcohol until a maximum a m o u n t of alcohol-soluble nitrogen products h a d been extracted, solutions containing from 40 per cent t o 60 per cent alcohol gave identical results. F r o m 6 j per cent t o 9 j per cent b y volume, t h e per cent of nitrogen products decreased rapidly. Osborne a n d Voorhees in their work used a n alcohol of 0.90 sp. gr. Later S h u t t 8 a n d Hummelg observed between t h e range of 60 per cent t o ;j per cent alcohol, b y weight, t h a t t h e nitrogen content of flour soluble in alcohol decreased with t h e increased strength of alcohol. Between 6 0 per cent a n d 86.4 per cent alcohol, b y weight, S h u t t observed t h e same order of solubility: with 86.4 p e r cent alcohol only 0 . I 2 per cent nitrogen; compared with 6 0 per cent alcohol 0.94 per cent nitrogen was noted. Hoagland,lO working with two samples of flour, observed t h a t more nitrogen was extracted with water t h a n with alcohol of strengths ranging from I O t o 2 0 per cent by weight. Increasing t h e strength t o 1
263. 2 3
4 5
6
A g r . Gazette X. S. Wales, Sept., 1896. J o u r . A m e r . Chem. Soc., 1900, Ibid.. 1905, 1068. Compt. rend., 1896, 123, 155. J o u r . f . prakt. Chem., Bd. 61, 344 (1854). Berichte d . L a n d w . Versuchs. Rostock, 1894. C o m p t . rend., 1896, 327. A r k . Bull. h-0. 53. U. S. Dept. of Agr., Bur. of Chem., Butt. h-o. 81. Ibid., Bull. No. 90. Central Exp. Farm, Ottawa, Can., 1907. U. S. Dept. of Agr., Bur. of Chem., Bull. S o . 105. THISJOURNAL. 3, No. 1 1 , 838.
Vol. 5 , NO.
11
7 j per cent b y weight, Hoagland found t h a t alcohol of 40 t o 4 j per cent by weight extracted as much or more nitrogen products t h a n strengths ranging between 60 t o 7 j per cent b y weight. He believes a 50 per cent alcohol b y weight t o be t h e logical strength t o use. It should be remembered t h a t Osborne a n d Voorhees separated t h e gliadin or its nitrogen from t h e alcohol-soluble nitrogen. T h e fact t h a t larger quantities of nitrogen-carrying bodies are extracted with weaker alcohols does not prove t h a t larger quantities of gliadin are extracted, yet nitrogen determinations are made a n d i t is assumed t h a t such is t h e case. T h e observations of Kjeldahl,’ lIathewson,* a n d Osborne a n d Harris3 showed t h e specific rotation of gliadin, [aID-92 t o be uniformly constant. Snyder4 introduced a method for t h e estimation of gliadin which proved unsatisfactory in t h e hands of others. Thatcher5 was unable t o use t h e method on soft wheat flours. Shaw a n d Gaumitz6 s t a t e in their paper t h a t “ t h e gliadin nitrogen should be corrected for t h e amide nitrogen present, b u t for most practical purposes this may be neglected,” especially “with old a n d unsound flours a correction must be made for t h e soluble amide bodies.” Chamberlain also is of t h e opinion t h a t amide compounds soluble in alcohol are considered as gliadin in t h e proposed methods. Greaves,’ in his thesis work, with t h e aid of t h e polariscope, found t h a t ;o per cent alcohol extracted more nitrogen in smaller quantities of flour t h a n where t h e same quantity of alcohol acted upon larger quantities of flour. Chamberlain recommended t h a t a t least IOO cc. of alcohol per 2 grams of flour should be used. I n working with alcohol ranging from 60 per cent t o 80 per cent b y weight, Greaves obtained t h e largest a m o u n t of nitrogen, with a few exceptions, with 7 0 per cent alcohol. T h e highest specific rotation [ a ] -89.80 was observed with 74 per cent alcohol, a n d he is of t h e opinion t h a t this is more nearly t r u e gliadin t h a n gliadin extractions made with other strengths of alcohol. I n studies with hot alcohol compared with cold, Chamberlain8 obtained more gliadin nitrogen in cold alcohol t h a n in case of hot alcohol. Leach9 recommends IOO cc. of ;j per cent hot alcohol per g r a m of material. Hoagland found t h a t a t a temperature of 7 j 0 C., a n d below t h e latter temperature, there is practically no temperature effect on the amount of nitrogen extracted. Greaves, employing tightly stoppered pressure flasks, obtained higher results with hot alcohol compared with cold. I n t h e earlier investigations on gliadin t h e experimental work was directed towards t h e preparation of pure gliadin, so t h a t i t was possible t o properly identify a n d classify it. I n t h e latter researches, when quantitative methods mere desired, t h e s u m total nitrogen 1 2
3 4
5
6
Centralbl. Agr. C h e m , 25, 197 (1896). J o u r . A m e r . Chem. Soc., 28, 1482 (1906) Ibid., 24, 844 (1903). I b i d . , 26, 263 (1904). I h i d . . 29, 910 (1907). Calif Bull., No. 212. J . Bioi. Chem., 9 , 3-4, 271.
Jour. A m e r . Chem. SOL..28, No. 1 1 . Food Inspection and Analyses, p. 232.
extracted by alcohol has been considered pure gliadin. carry n-ith i t t h e coagulable a n d non-coagulable I n literature, there is nothing t o show t h a t all of t h e nitrogen which was removed by t h e salt solution. Sitrogen determinations were made by the straight nitrogen compounds present in t h e alcohol-soluble is t h e nitrogen of pure gliadin. There is, on t h e other Kjeldahl method in t h e flour, the direct extraction of hand, some indication t h a t a p a r t a t least of t h e t h e flour with io per cent alcohol, t h e coagulum in nitrogen products is a p a r t of less complex substances this alcohol-soluble, t h e alcohol-soluble resulting after t h a n gliadin, e. g . , amides. There is some evidence t h e extraction of t h e flour with I per cent salt solution, t h a t substances closely allied t o gliadin are present t h e salt-soluble extraction a n d t h e salt-soluble insoluble a n d which O’Brien’ believes “closely merge into one in 7 0 per cent alcohol. T h e results obtained for t h e above determinations are recorded in Table I. another.” T h e general methods adopted for t h e separation of T.4BI.E 1 -ALCOHOL-SOLUBLE P R O T E I S S BY D I R E C T , IXDIRECT AND C O A G U gliadin from its solvent have been either t o dilute t h e LATION METHODS alcohol a n d a d d salt solution or increase t h e strength of alcohol b y adding absolute alcohol. Osborne states t h a t distilling off t h e alcohol in vacuo causes t h e .-i s settling out of gliadin. This latter method has been .made use of b y t h e writer when handling large quantities of material. B u t where t h e quantity of material is small a n d t h e exact purity of t h e material is of secondary importance, t h e writer has evaporated off t h e alcohol in t h e open, thereby causing precipitation or settling o u t of pro0 82 1 1 4i 0 61 0 40 0 25 0 35 0 60 2 2 Oi 1 11 0 ;3 0 45 0 29 tein. Owing t o t h e peculiar properties of gliadin 0 52 0 81 3 2 18 1 22 0 8i 0 47 0 32 0 61 0 91 a n d t h e similar properties of t h e coagulum i t is reason1 6 1 4 2 73 1 12 0 61 0 33 0 84 1 li able t o believe t h a t t h e y are one a n d t h e same substance. T h e gliadin, however, is purer t h a n t h e preThe d a t a given in Table I give results by t h e direct cipitate due t o t h e fact t h a t it is redissolved a n d pre- process of extracting flour for nitrogen-carrying cipitated a number of times. T h e nitrogen bodies bodies as well as t h e reverse process. T h e results dissolved b y t h e alcohol a n d not precipitated are non- obtained are not comparable with each other in t h e gliadin bodies which from a quantitative point of view two methods. More t h a n this, t h e d a t a show t h a t are generally included as gliadin nitrogen. salt extracts a part of t h e alcohol-soluble nitrogenThis means of separating t h e alcohol-soluble nitrogen carrying bodies a n d ;o per cent alcohol b y volume materials into t w o groups offers a satisfactory working applied directly t o a flour removes larger amounts of basis for t h e estimation of gliadin in flour or gluten. protein t h a n can be accounted for b y t h e indirect I n some respects such a method is similar t o t h e one method. T h e excess amount of alcohol-soluble b y t h e direct method possibly has been altered upon e r a p o r a t in vogue for t h e estimation of casein in milk. I n order t o learn whether or no i t was possible t o ing t h e salt-soluble a n d hence is n o longer soluble in make use of t h e above method for t h e estimation of this strength of alcohol. T h e d a t a further show t h a t gliadin i t was necessary t o include with i t t h e estima- a large p a r t of t h e alcohol-soluble nitrogen-carrying tions for t h e salt-soluble protein substances found in bodies are coagulated when t h e alcohol is removed a n d boiled with water. These results further indicate flour. I n t h e preliminary experiments a series of four flours t h a t a p a r t of t h e alcohol-soluble nitrogen-carrying were selected. These were first extracted with one bodies is estimated as leucosin a n d edestin when I per cent salt solution ( S a C l ) , followed by treating per cent salt solution is used. Similar results were t h e insoluble residue with sufficient 9 j per cent alcohol obtained b y Chamberlain who extracted t h e flour t o make a n alcohol 7 0 per cent b y volume. A direct with alcohol followed b y extracting t h e alcohol residue io per cent alcohol separation was also made. Besides with a j per cent solution of potassium sulfate. The this, jo C C . aliquot of t h e direct alcohol-soluble separa- amount of nitrogen-carrying bodies extracted from t h e tion was evaporated t o within j cc., j o cc. of water alcohol residue as obtained by him was only one-fifth added, boiled, a n d t h e n cooled t o room temperature. as much as t h a t obtained by t h e direct extraction of t h e A heavy coagulum or settling o u t resulted upon t h e flour with j per cent potassium sulfate. Perhaps t h e most interesting results obtained were evaporation of t h e alcohol a n d increased in amount t h e remarkably close agreement betn-een t h e nitrogen upon cooling after t h e boiling. An aliquot of t h e I per cent salt-soluble was evapora- in t h e alcohol-soluble coagulum a n d t h a t of t h e alcoholted t o dryness a n d t h e n extracted with 70 per cent soluble nitrogen where t h e flour h a d first been extracted alcohol, in order t o remove t h e gliadin which, ac- with I per cent salt solution together with t h a t excording t o Teller a n d confirmed b y Chamberlain. is tracted in t h e direct salt-soluble. These d a t a add appreciably soluble in I per cent salt solution. T h e further evidence t h a t alcohol extracts other nitrogenocs residue remaining after t h e alcohol extraction should substances from flour t h a n gliadin and t h a t “gliadin be composed largely, if not all, of edestin a n d leucosin. is appreciably soluble in I per cent salt solution.” T h e alcohol extraction in t h e dried salt solution should I n this series of experiments approximately 7 2 per cent of t h e total alcohol-soluble nitrogen is gliadin nitrogen 1 Ann& of Bol., 1895, p. 182 i
T H E J O r R N A L O F IiVD L'S T R I A L AAVD E,VGILVEERING C H E M I S T R Y
920
T'ol.
j,
No.
11
based on either t h e coagulum nitrogen or t h e alcoholsoluble nitrogen b y t h e indirect method. Owing t o t h e close agreement in t h e nitrogen cont e n t in t h e coagulum compared with t h e nitrogen content in t h e alcohol-soluble b y t h e indirect method i t appears reasonable t o t h e writer t h a t either one of t h e t w o methods mentioned here would be satisfactory for t h e estimation of gliadin nitrogen. T h e method for t h e determination of t h e coagulable nitrogen is t h e shortest a n d t h e better one t o follow of t h e t w o a n d as a result t h e writer has devoted his time t o a s t u d y of this method. As h a s been s t a t e d , t h e strength of alcohol best suited for gliadin estimation has never been fixed. Greaves believes t h a t a 54 per cent alcohol is t h e proper strength t o use in polariscopic estimations, while variable strengths have been suggested i n gravimetric estimations. F o r this reason i t is necess a r y t o determine a strength t h a t would be suitable for t h e work involved. T w o samples of flours were digested with cold 4 j , 50, jj, 60, 6 j , j o , jj, 80, a n d 8 j per cent alcohol b y volume a n d t h e nitrogen determined in a n aliquot of t h e alcohol-soluble as well as in t h e coagulum resulting from another aliquot of t h e alcohol-soluble. Nitrogen 'determinations were also made in t h e phosphotungstic precipitates resulting from t h e coagulum filtrates. T h e results for nitrogen in t h e alcohol-soluble, t h e coagulum filtrate a n d t h e s u m of t h e last t w o determinations are recorded in Table I1 of this paper.
results agree with those observed, first b y Teller, a n d later b y other investigators. F r o m these results i t appears t h a t a jo per cent alcohol b y volume is satisfactory t o use for t h e estimation of t h e per cent of nitrogen in t h e coagulum. T h e slight variations noted in t h e totals compared with t h e results b y t h e direct method are undoubtedly due t o analytical error. T h e following method was finally adopted for t h e s t u d y of coagulum fraction of t h e alcohol-soluble. Four grams of flour were digested with zoo cc. of j o per cent alcohol b y volume, shaking t h e entire contents at intervals of five minutes a p a r t during t h e first t w o hours, t h e n allowing t o s t a n d twenty-four hours followed b y filtering clear. iZliquots of j o cc. each were t a k e n from t h e thoroughly mixed filtrate, e r a p o r a t e d slowly t o within 5 cc. volume, j O cc. of water a d d e d t o each a n d further evaporated a t a temperature near t o boiling until contents of beakers approximately amounted t o I O cc., repeating j o cc. of water were added a n d again evaporated down t o within 3 j cc. T h e beakers were t h e n removed, 2 j cc. water added, allowed t o cool t o room temperature (21') a n d finally filtered. I n case of turbid filtrates, t h e filtrates were repeatedly filtered until clear. T h e coagulum is difficult t o remove in most instances, owing t o t h e sticky nature of this substance, a n d i n such cases t h e mass was gathered b y t h e use of steel spatula a n d policeman a n d transferred t o filter paper, occasionally using cold, distilled water t o facilitate t h e transferring a n d washing of t h e coagulum. S i t r o g e n determinaTABLE 11-AMOUNT OF COAGULABLE NITROGEN OBTAINEDI N DIFFERENT tions were made according t o t h e Kjeldahl method. STREh.CTHS O F ALCOHOL ~Lc S i Z Z s ~ 2 4 T h e~ proper corrections for paper a n d reagents were t a k e n into consideration in all determinations for .E .s .o .e .E .e .e .E .e nitrogen. T h e filtrates resulting from t h e separation of t h e coagulum were treated with sufficient phosphotungstic acid a n d nitrogen determined i n precipitate a n d filter paper. A. D i r e c t . . . , , 1.2,3 1 . 2 9 1 . 3 0 1 . 3 0 1 . 2 5 1 . 2 1 0 . 9 9 0 . 7 8 0 . 3 3 Coagulum, 0.87 0.82 0 . 8 4 0.84 0.77 0.75 0.61 0.59 0.15 I n a series of experiments comprising sixty different Precipitated flours obtained from different sources, representing filtrate.. . 0 . 3 9 0 . 4 5 0 . 4 2 0 . 4 2 0 . 4 1 0 . 3 9 0 . 3 2 0 . 2 3 0 . 1 4 Total. . . . . . , 1 . 2 6 1 . 2 7 1 . 2 6 1 . 2 6 1 . 1 8 1 . 1 4 0 . 9 3 0 . 8 2 0 . 2 9 different climatic zones in t h e United States, some B. Direct. . . . . 1 . 3 6 1 . 6 3 1 . 6 3 1 . 6 2 1 . 5 7 1 . 4 5 1 . 0 5 0 . 8 6 0 . 1 6 of which known as spring or winter wheat patents, Coagulum., 1 . 1 4 1 . 1 8 * 1 . 2 1 1.21 1 . 1 7 1 . 0 8 0 . i 2 0 . 5 0 0 . 0 2 others straight, or bakers, some of better baking Precipitated filtrate. . . 0 . 2 4 0 . 3 7 0 . 3 4 0 . 3 5 0 . 3 5 0 . 3 1 0 . 3 6 0 . 2 6 0 . 1 3 qualities t h a n others, were subjected t o t h e above Total, . . . . . . 1 . 3 8 1 . 5 5 1 . 5 5 1 . 5 6 1 . 5 2 1 . 3 9 1 . 0 8 0 . 7 8 0 . 1 5 mentioned method for t h e determination of gliadin. T h e average maximum a n d minimum results for There are several things of interest t o be noted in ,
t h e d a t a given in Table 11. T h e per cent of nitrogen obtained in t h e coagulum after removing t h e alcohol runs quite uniformly regardless of strengths of alcohol ranging between so per cent t o 6 j per cent inclusive alcohol b y volume. F r o m 7 0 per cent there is a marked decrease in t h e per cent of nitrogen until a t 8 j per cent only a b o u t 2 j per cent of t h e total nitrogen in t h e alcohol-soluble is obtained. T h e per cent of nitrogen in t h e phosphotungstic acid precipitated filtrate bears a similar relation. T h e s u m of t h e per cents of nitrogen in t h e coagulum a n d phosphotungstic acid precipitate agree fairly well with t h e per cents of nitrogen obtained b y t h e direct alcohol extraction. I n regard t o t h e per cents of nitrogen-solubles in different per cents of alcohol i t will be noted t h a t t h e
TABLE111-PER CENTNITROCEA I N ALCOHOL FRACTIOYS
Average (60) ... , 1 . 9 ; Maximum . . . . _ 2 . 7 2 Minimum... . . , 1.31
1.105 1.560
0.610
0.755 0.295 1.210 0.440 0.440 0.120
0.033 0.19 0.09
38.32 50.0 31.0
68.32 86.40 50.50
per cents of total nitrogen in flour, alcohol-soluble nitrogen, coagulable nitrogen i n alcohol-soluble a n d nitrogen in phosphotungstic acid precipitate are recorded in Table 111. I n addition t h e per cent of coagulable nitrogen expressed in per cent of t o t a l nitrogen in t h e flour a n d alcohol-soluble are included.
XOV.,
I913
T H E J 0 l i R ; V A L O F I - V D C S T R I A L AAVD E S G I S E E R I N G C H E M T S T R Y
According t o t h e results summarized in Table 111, it will be noted t h a t t h e per cent of coagulable nitrogen of total nitrogen in t h e flour is 38.52, while t h e per cent of coagulable of total alcohol-soluble is 68.32. I t is further noted t h a t all of t h e alcohol-soluble nitrogen is not coagulable nitrogen a n d t h a t which is not coagulable is precipitated b y phosphotungstic acid. If t h e coagulable nitrogen contains all of t h e gliadin nitrogen of flour, i t will be noted t h a t this nitrogen does not bear a n y relation t o t h e t o t a l alcohol-soluble, nor total nitrogen of t h e flour. I n connection with this work i t was t h o u g h t desirable t o carry o u t a few comparative tests as t o t h e purity of t h e coagulum from a nitrogen point of view with t h e mass obtained i n t h e alcohol-soluble. Four flours from as m a n y different states were selected for this work. T h e per cent of total solids, per cent of nitrogen in t h e solids, t h e per cent coagulum, per cent of nitrogen i n coagulum, per cent ash in coagulum, a n d per cent of sugar in filtrate resulting from t h e separation of t h e coagulum are recorded in Table I\’. TABLEIV-PURITY
OF
ALCOHOL-SOLUBLE COMPAREDWITH PURITYOF COAGULUM
Kansas . . . . . . . . . . . . . 10.11 12.02 Washington.. . . . . . . 11.10 New I‘ork . . . . . . . . . . 1 0 . 5 0
So. D a k o t a . . . . . . . . .
8.61 9.90 8.47 5.14
4.31 5.98 6.12 5.18
13.69 14.88 12.09 10.42
0.11 0.12 0.23 0.39
0.121 0.058 0.266 0.244
T h e degree of purity of t h e alcohol-soluble based upon t h e nitrogen content a n d total solids is approximately less t h a n one-half t h e purity of pure gliadin TABLE \‘-PER
NO.
O.C. ..........................
CENT CO.4CULABLE
Per cent N in gluten 13.66
R . W . B. . . . . . . . . . .
B.L. . . . . . . . . . . . . . . . P.............................
12.96
....................... 14.24 . . . . . . . . . . . . . . . . . . . . . . . 14.28
SITROCEN IN
1
T i m JOURNAL, 4, S o . 3 (1912).
of preparing gluten in dry form which when moistened with water takes on all of t h e characteristics of t h e gluten from which i t was prepared. I t occurred t o t h e writer t h a t i t might be well t o t r y t h e above method for t h e estimation of gliadin on prepared glutens obtained from flours in which t h e method h a d been tried directly. Accordingly, one-half g r a m lots of I O X X bolted gluten from six different flours were digested with t w o hundred centimeters of j o per cent alcohol b y volume a n d t h e method as described above was followed from this point. T h e per cent of nitrogen in dried gluten, t h e per cent nitrogen in t h e alcoholsoluble, t h e per cent nitrogen in coagulable of t h e alcohol-soluble, t h e per cent nitrogen i n t h e phosphotungstic acid precipitated filtrate, t h e error in t h e determinations, a n d t h e a m o u n t of gluten in I O O grams of flour from which d a t a t h e grams of nitrogen in t h e gluten, t h e alcohol-soluble a n d coagulable, have been calculated a n d are recorded in Table V. T h e per cent of coagulable nitrogen as found in t h e alcohol-soluble extract of t h e flour is introduced in t h e last column for comparison. When t h e possible variations in t h e results for t h e determinations of d r y gluten, t h e solubility of gliadin i n t h e process of washing t h e gluten out of t h e flour, as well as t h e possible error involved in making nitrogen determinations, are considered i t will be noted (Table 1‘) t h a t t h e per cent coagulable nitrogen found in alcohol-soluble of t h e gluten compares remarkably closely with t h e coagulable nitrogen obtained b y t h e direct extraction of t h e flour with alcohol. I n t w o instances, aiz., B. L. a n d SI. 15, t h e error is somewhat large, b u t on closer s t u d y i t will be further noted t h a t t h e results obtained i n t h e gluten r u n lower i n all instances GLUTENA N D CALCULATED
Per Per cent hT in cent X Per phosphotungalcohol- r e n t K in stic presoluble Coagulable cipitate 6.64 6.32 0.46 8.56 5.76 2.68 6.64 4.96 1.60 6.88 4.88 2.04 i 52 6.56 0.96 6 48 4.96 1.50
(17.6 per cent S ) a n d for t h e coagulum t h e nitrogen content (av. 1 2 . 7 j ) agrees with t h e nitrogen content of gluten ( 1 2 . j f per cent S).l T h e per cent of sugar found a n d t h e per cent of ash are small a n d seemingly do not influence t h e purity of t h e proteins t o a n y great extent. J u s t how much f a t t h e coagulum carries was not determined, b u t from determinations made in non-ether extracted preparations of gliadin sometimes as high as 13.5 per cent of ether extract was found a n d presumably t h e larger a m o u n t of f a t in t h e coagulating method includes fat in t h e coagulum which f a t , if included in gliadin. XT-oald reduce t h e nitrogen per cent in gliadin t o figures practically as low as those obtained b y t h e writer for t h e coagulum. I n research bearing on t h e properties of gluten a n d i t s effect upon the baking qualities of flour, we have been exceedingly f o r t u n a t e in working o u t a method
921
Error
t o . 12 + O . 12 -0.08 f0.04 0.00 -0.02
IN
PER CENT OF FLOUR
Per cent gluten in flour 14.20 9.56 9.35 1T.20 11.60 12,iO
Per Per cent Per cent alcohol- cent coagu- Per cent gluten hT soluble in lable N in coagulable per cent found in per cent per cent of flour in flour of flour of flour 0.90 0.96 1,95 0.94 1.33 0.82 0.55 0.56 0.46 0.58 1.34 0.56 0.84 0.92 2.23 1.18 1.65 0.87 0.76 0.69 1.81 0.82 0.63 0.74
except one t h a n was t h e case in t h e determination in t h e flour. A possible explanation for t h e lower results other t h a n those mentioned above m a y be either t h a t t h e total alcohol-soluble material contained in t h e gluten was not entirely extracted or t h e alcohol-soluble obtained in flour contained other proteins which coagulate with t h e coagulum a n d do not combine with t h e glutenin t o form gluten. &Ita n y rate, gluten prepared in t h e manner described is excellently suited for t h e preparations of t h e component proteins of gluten in pure form. T h e ether-soluble materials can be removed first, then t h e alcohol-soluble. etc. I t is a recognized fact t h a t gliadin is slightly soluble in n-ater, somewhat in I per cent salt solution a n d practically insoluble in I O per cent salt solution. Xccordingly, one mould expect t o find a p a r t of t h e gliadin in solution i n t h e filtrates resulting from t h e
T H E J O C R S A L O F I , V D l ' S T R I A L A S D E-VGINEERI,VG C H E M I S T R Y
922
separation of t h e coagulum of the alcohol-soluble. I n t h e writer's experience i t has been found t h a t t h e coagulum is difficultly soluble in cold water. Further, upon evaporating t h e filtrates very small quantities of coagulum result which when transformed into t h e coagulable s t a t e remain practically insoluble. T h e a m o u n t of coagulable material which passes into t h e filtrates must be exceedingly small a n d in no case has t h e writer obtained sufficiently large quantities t o warrant a determination to be made. F r o m a quantitative point of view, i t is safe t o assume t h a t practically all of t h e gliadin nitrogen has settled out or coagulated a n d t h e nitrogen bodies present in t h e filtrates are of less complex form. Owing t o t h e difficulties met with in transferring t h e sticky coagulum formed in t h e method described above a n d since t h e nitrogen contained i n t h e coagulum, together with t h a t found in t h e filtrate, equal t h e nitrogen found in t h e alcohol solution, t h e writer has deemed it a better method of procedure t o estimate t h e nitrogen in t h e filtrate resulting from t h e separation of t h e coagulum a n d deduct this result from t h e nitrogen found in t h e alcohol-soluble extraction t o get t h e gliadin nitrogen contained in t h e flour. The method for t h e estimation of gliadin nitrogen would t h e n read as follows: Digest four grams of flour with z o o cc. of j o per cent alcohol b y volume. Shake t h e contents at five-minute intervals for t h e first t w o hours. t h e n let s t a n d over night a n d finally filter clear. Determine t h e per cent of nitrogen in z j cc. aliquots of t h e alcohol-soluble. Evaporate slowly 5 0 cc. aliquots of t h e alcohol-soluble t o within j cc. volume, a d d j o cc. distilled water, bring near t o boil, a n d continue process until volume has been approximately reduced t o I O cc. Repeating, a d d 50 cc. of water a n d boil down t o within 3 5 CC. volume. Allow contents of beakers t o cool t o room temperature, t h e n filter, If, necessary, repeat filter until filtrate becomes practically clear. Estimate t h e per cent of nitrogen contained in t h e filtrate a n d deduct this result from t h e per cent of alcohol-soluble nitrogen t o get t h e per cent of gliadin nitrogen in t h e flour. CONCLUSlOSS
I. Alcohol
solutions extract two groups of nitrogen-carrying bodies in wheat flour a n d gluten. Upon evaporating off t h e alcohol followed b y t h e addition of water, one of t h e groups, aiz., gliadin, separates o u t , t h e other remaining i n solution. 11. Salt solution of I per cent strength extracts a p a r t of t h e gliadin contained in viheat flour. 111. T h e gliadin nitrogen obtained b y t h e indirect method is much lower t h a n t h a t obtained b y t h e direct method, b u t agrees remarkably closely with t h e results obtained for nitrogen in t h e coagulum in t h e alcoholsoluble. IT'. Of t h e two methods for t h e estimation of gliadin, t h e coagulation method is t h e shorter a n d is more suitable t o use for this reason t h a n t h e indirect method. T'. Alcohols ranging between jo per cent t o 6 j per cent, inclusive, b y volume, extract equivalent a m o u n t s of coagulum nitrogen. Above 6 j per cent
N o v . , No.
11
alcohol b y volume there is a rapid falling off i n t h e a m o u n t of coagulum nitrogen obtainable. Alcohol of j o per cent by volume has been adopted b y t h e writer for t h e determination of gliadin nitrogen. 1.1. About 6 8 per cent of t h e total alcohol-soluble nitrogen is coagulable. T'II. Alcohol-soluble, incoagulable nitrogen is precipitated b y phosphotungstic acid. This, together with t h e coagulable nitrogen, equals t h e total alcoholsoluble nitrogen. VIII. In t h e same flours as much coagulable nitrogen is obtained in specially prepared gluten as in case of t h e flour. I X . T h e easiest a n d shortest method for t h e estimation of t h e gliadin in flour is t o estimate t h e nitrogen i n t h e uncoagulable portion of t h e alcoholsoluble a n d deduct this result from t h e total alcoholsoluble nitrogen. L~ASHISGTON E X P E R I M E NSTT A T I O N PULLMAN
QUALITY OF THE MASSACHUSETTS MJLK SUPPLY A S SHOWN BY THE INSPECTION OF THE STATE BOARD OF HEALTH B y HERMAN C. L Y T H C O E
Received August 14, L913
During t h e past year a n investigation of t h e milk supply was undertaken with special reference t o certain enzyme a n d other reactions which could possibly differentiate raw milk from pasteurized milk a n d new milk from old milk. T h e n a t u r e of these reactions a n d t h e source of t h e substances causing t h e m are not yet clearly settled, b u t a s t u d y of t h e literature shows t h e following reactions which m a y be of a n enzyme nature: R a w milk contains a diastase capable of hydrolyzing 0.01 t o 0 . 0 2 per cent of starch, a catalase which will liberate oxygen from hydrogen dioxide, a peroxidase which will cause hydrogen dioxide t o react with certain organic substances, thereby producing colors, a n d reductases capable of decolorizing methylene blue solutions. Pasteurization has more or less influence upon t h e above reactions depending upon t h e temperature of pasteurization a n d t h e length of time subjected t o this temperature, a n d t h e reactions are also modified b y t h e age of t h e milk. T h e reductases are produced b y bacteria according t o Konning,' Seligman,2 a n d Grimmer.3 On t h e cont r a r y Seligmanz states t h a t some reductase m a y possibly exist in milk as a n enzyme. Romer a n d Samesl question t h e enzyme nature of reductase a n d s t a t e t h a t i t is produced b y t h e destruction of t h e cells of t h e m a m m a r y glands during milking, as t h e first milkings have b u t slight reducing properties a n d t h e last milkings are highly reducing. Same@ is opposed t o t h e enzyme nature of reductase a n d thinks t h a t t h e Seligman assumption t h a t t h e reduction is due t o bacteria is too far reaching. Salus6 .\filckviutschl.
2
Z . Hyg.,68, 1. .Ililckw~irlsckl. Zenlr., 6, 243. Z . S a k r . Genussm., 20, 1.
3
.
Zenlv.. 4 , 156.
1
4 6
6
Jlil6ku,irtschl. Zenlv., 6, 462. ..lvck. Hyg., 75, 371.
