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 ENGINEERIAVG C H E M I S T R Y t h e flavor of these beverages is quite up t o the standa r d of fresh juice preserved a s above indicated, b u t it is still quite close t o t h e normal taste of orange juice, if t h e evaporation has been carefully done. Of course, t h e vacuum treatment removes t h e flavoring materials from t h e juice, and it is necessary t o add oil for flavoring purposes, better a t t h e time of making t h e beverage t h a n before pasteurization in the vessel. The concentrated juice will keep under t h e same conditions as t h e fresh, i . e., after pasteurization in the absence of free oxygen; t h e dried material cannot be pasteurized, as it fuses together a t pasteurizing temperatures, b u t pasteurization is not necessary, provided water and air are excluded. There is a considerable literature, especially of patents, regarding t h e preservation of fruit juices, b u t it is thought inexpedient t o include a n y of these references in this abstract. T h e recovery of t h e flavoring oils from t h e peels of citrus fruits is at present carried on mainly b y rather crude methods in Italy and Sicily, under conditions of cheap labor with which American packers cannot compete. A process €or recovering this valuable oil from t h e peels of Florida oranges must therefore be one which will handle a large number of peels a t very little cost. 1-arious mechanical methods of pressing, rolling, abrading, etc., were tried without much promise of success. Soaking methods, in which t h e ground peel is covered with water, t o t h e surface of which t h e oil rises and is there drawn off, gave low yields of fair quality oil, b u t while simple, these processes are rather inefficient. After considerable experimentation it was found that a very satisfactory oil could be produced by grinding t h e peel, submitting t h e ground material t o a current of water vapor a t greatly reduced pressure and condensing a n d separating t h e oil. Ordinary Florida oranges yielded about 0 . j cc. of oil per peel, while t h e late Valencias gave from I t o I . 5 cc. per peel. T h e liberation of t h e oil appears t o be favored by previous partial drying of the peel. The oil obtained from Florida oranges as above indicated, has been used repeatedly in cakes and candies, when dissolved in alcohol, and has given excellent results. I t appears t o be u p t o the requirements of the existing legislation. As is well known, t h e flavoring oils from citrus fruits rapidly deteriorate on exposure t o t h e air, especially in t h e light, acquiring a very offensive turpentine-like odor. I t was found during this research t h a t this could be obviated b y t h e addition t o t h e oil of about I O per cent b y volume of absolute alcohol; oil so treated was allowed t o stand exposed, t o diffused daylight at room temperatures for many months, without deterioration. It is not t o be recommended, however, t h a t such oils be kept under these conditions; keeping in the dark in a cool place is far better, even with protecting agents present. T h e use of 1.5 t o z per cent of olive oil has also been recommended for this purpose; this gives some protection, b u t is not as good as t h e alcohol. Sealing in an atmosphere of carbon dioxide is also effective in protect-
1701. 8, S o .
2
ing the oil for a long time. On t h e whole, however, the addition of absolute alcohol gave the best results, and it seems t h a t it would be well t o admit of this treatment of such oils for their preservation in t h e United States Pharmacopoeia and other standards, The recovery of the citric acid from the juice of Florida citrus fruits (for t h e sake of t h e acid alone) is scarcely worth while, in view of t h e small amount available, rarely over 0 . 7 per cent in the orange, or I , 5 per cent in t h e grapefruit. 4 s a matter of scientific interest, however, it was found t h a t Wehmer’s Citi,omyces molds would grow on t h e sterilized juice in t h e presence of calcium carbonate, and convert a considerable proportion of t h e residual sugar into citric acid. The commercial application of such a process seems rather hypothetical. From t h e results of this investigation it appears, then, t h a t t h e preservation of t h e juice of the orange and grapefruit is practicable, t h e method depending on pasteurization out of contact with air, and t h a t the recovery of the flavoring oil from t h e peels m a y be accomplished commercially b y methods of vacuum distillation, b u t t h a t t h e recovery of t h e citric acid for itself alone is not practicable. Since this abstract was prepared, a valuable and interesting paper b y Will1 has appeared, relating t o t h e utilization of these culls in the California citrus industry. MELLOS IXSTITGTE O F IXDUSTRIAL RESEARCH CNIVERSITY OF PITTSBURGH. PITTSBURGH, Pa.
ON THE CHEMICAL CONSTITUTION OF THE PROTEINS OF WHEAT FLOUR AND ITS RELATION TO BAKING STRENGTH By M. J. BUSH Received June i, 1915
IKTRODUCTION
The most generally accepted definition of “baking strength” of a wheat flour is t h a t p u t forward b y Humphries and Biffen,2 in 1907: which states t h a t a “strong wheat is one which yields flour capable of making large, well-piled loaves;” a definition similar t o t h a t of jag^,^ who states t h a t “strength. . . . defined as t h e measure of the capacity of t h e flour for producing a bold, large-volumed, well-risen loaf.” Since the value of wheat (other things being equal) depends on t h e so-called “strength” of t h e flour which may be made from i t , it is obviously of great importance t h a t complete knowledge be obtained concerning t h e factors which cause strength, and t o this end a n enormous amount of scientific work has been done, especially during t h e last twenty years. I n spite of the fact t h a t some of the foremost investigators of t h e world have bent their energies t o this task, t h e problem is not yet completely solved, although considerable light has been thrown on t h e subject. It is not yet possible t o correlate baking strength with a n y chemical or physical factor t o such an extent t h a t a simple laboratory test or group of tests mill always 1
2
THISJOURNAL, 8 (1916). 78-86. “The Improvement of English \%‘heat,’’ Jour. A I Y . Sci.. 2 (1907).
1-16. a “Technology of Bread Making,” Chap. XV, p . 291, 1911.
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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
furnish a n infallible guide, but it is necessary t o mill the wheat into flour a n d have a sample of it actually baked into a loaf of bread b y an,expert baker, before its strength can be accurately ascertained. FACTORS
WHICH
MAY
INFLUENCE
BAKING
STRENGTH
Almost every known constituent, or group of constituents, a n d almost every known physical a n d chemical property of flour has been investigated with respect t o its possible relation t o baking strength, but as yet no one is believed t o have discovered a limiting factor or group of factors which completely solves the problem. Moreover, there is a general disagreement among many of the leading investigators as t o t h e importance which should be attached t o each factor or set of factors, a n d two workers frequently have arrived a t exactly opposite conclusions after having investigated practically the same problem; however, much of this confusion is caused b y the use of different methods of analysis. A brief review of some of the more important work which has been done will serve t o bear out t h e preceding statement, as well as t o indicate t h e many sides from which the question of flour strength has been studied. GLIADIN-GLUTENIN RATIO-AS soon as Osborne a n d Voorhees,’ in 1893, established t h e composition a n d properties of t h e wheat proteins, attention was a t tracted t o gliadin a n d glutenin, the two conspicuous a n d characteristic proteins of wheat, which. were shown t o make u p the gluten, the more or less elastic binding material which enables flour t o be made into t h e dough with its characteristic elastic, gas-retaining property, a n d which may be separated from t h e starch a n d soluble proteins by the well-known process of washing the dough in a stream of water. Fleurent,2 in 1896, claimed t h a t flour strength depends on t h e proportion of gliadin t o glutenin present in the gluten of t h e flour. He concluded from his experiments t h a t t h e optimum ratio was 75 parts of gliadin t o 2 5 of glutenin or 3:1. He assigned certain limits, outside of which flours were said t o be of poor baking quality. Snyder,3 in 1899, published similar results, although he fixed his ideal ratio at 6j:3 j. He also states4 t h a t the quality rather t h a n t h e quantity of gluten is the important factor, because he was able t o a d d up t o 20 per cent starch t o flour without decreasing its baking quality. Regarding t h e quantity of gluten in flour, t h e amount of gliadin present, the ratio of gliadin t o glutenin, and t h e relation of these t o baking quality, it suffices t o say that the results of different investigators quite frequently are not concordant. However, as mentioned before, this is in a considerable measure due t o different analytical methods employed b y different workers. C R U D E GLUTEN-The crude gluten determination, which consists essentially of washing t h e gluten free 1 “ T h e Proteids of the Wheat Kernel,” Am. Chem. J , 15 (1893). 392-471: Ibid.. 16 (1894), 524-535. 2 “Sur une method chimique d’appreciation de la valeur boulangere des farines de bl6,” Comfit. rend., 123 (1896), 755-758. Mznn. Exfi. Sta. Bull., 62 (1899). 4 U. S. Dept. Agr., Bull 101 (1901).
I39
from starch a n d soluble material by means of water, a n d weighing the gluten, both in a wet and dry state, was for a long time considered of great value, b u t Snyder and Norton,l in 1906, Chamberlain,2 in 1906, a n d others showed t h a t it gave but little information which might not be gained from a determination of total nitrogen or alcohol-soluble nitrogen. Nevertheless, it is still used extensively b y millers a n d bakers, a n d in technical laboratories. P H Y S I C A L STATE O F G L U T E N A N D S U G A R CONTENT-
I n 1907, Wood3 published the results of a thorough a n d systematic study of the chemistry of flour strength. He concluded t h a t there is no difference in the chemical constitution of gliadin a n d glutenin from strong a n d weak flours, a n d decided t h a t strength (particularly shape of loaf) is much more closely related t o the physical state of gluten, which in t u r n is profoundly affected b y t h e presence of electrolytes. He showed t h a t minute quantities of acids and bases tend t o “disperse” gluten, making it weak and inelastic, while small quantities of neutral salts have t h e opposite a n d consequently beneficial effect. Furthermore, he found t h a t the volume of a loaf of bread is proportional t o the rate of carbon dioxide evolution resulting from diastatic activity of yeast in t h e later stages of fermentation. I n other words, he concludes t h a t loaf volume depends on t h e amount of available sugar in the later stages of fermentation. Alway a n d H a r t ~ e l l , ~ in 1909, however, performed experiments which led them t o say, in contrast t o Wood’s findings, “there is clearly no direct connection shown between the size of the loaf a n d t h e volume of gas evolved. The thirteen flours which gave the largest loaves evolved on t h e average somewhat less gas t h a n the other thirteen flours.” Shutt5 states t h a t from his experimental evidence he was unable t o find a n y relation between size of loaf and sugar content. ENZYMES-Comparatively less study has been made of t h e enzymes of flour and their relation t o strekgth. Perhaps t h e most prominent work in this field is t h a t which was done simultaneously but independently b y Baker and Hulton? a n d by Ford a n d Guthrie,’ in 1908. They point out t h a t both proteoclastic a n d amyloclastic enzymes are present in flour a n d in many instances may exert a profound influence on its breadmaking qualities. Baker a n d Hulton state t h a t “it is obvious t h a t the strength of a flour must be closely connected with the gluten, although no doubt the presence of enzymes, soluble carbohydrates, a n d mineral constituents all play a part.” Koch,* in 1914, found no difference in t h e quantity of diastase in strong a n d weak flours, after extracting them with “Crude Gluten,” J . A m Chem. Soc., 28 (1906), 8-25. “Properties of Wheat Proteins,” Ibid , 28 (1906), 1657-1667. 3 “ T h e Chemistry of Strength of Wheat Flour,” Jour. Agr. S c i , 2 (1907), 139-161 and 267-277. 4 Neb. Exp. Sta., 23rd Annual Report, 1909 “Flour-the Relationship of Composition t o Bread Making Value,” Canadzan Mzller and Cerealist, 5 (1913), 176-178. 6 “Conditions Affecting the Strength of Wheaten Flour,” Jour. SOL. Chem Ind.. 27 (1908), 368-376. 7 “ T h e Amylolytic and Proteolytic Ferments of Wheaten Flour and Relation t o Baking Value,” Jour. SOL Chem. Ind., 27 (IYOS), 389-393. 8 “ T h e Diastase and Invertase Content of Wheat Flour and Their Relation to BAking Strength,” Thesis for Master’s Degree, University of Minnesota, June, 1914. 1
2
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140
water a t o o according t o the method of Thatcher and Koch.‘ C O N C E N T R A T I O N O F H Y D R O G E N IOIW-H. JessenHansen,2 in 1911, finds a close relationship between t h e concentration in hydrogen ions a n d baking strength, a n d asserts t h a t there is a n optimum hydrogen-ion concentration for flour, t h e poorer flours having lower concentrations. He attributes t h e beneficial effects of neutral salts a n d “flour improvers” on flour t o t h e fact t h a t they raise t h e hydrogen ion concentration. SOLUBLE PROTEINS-There does not seem t o have been a very considerable amount of work done regarding t h e r6le of t h e soluble proteins as a factor in baking strength. Snyderja in 1897, says “When any of t h e wheat proteids except gliadin or glutenin are extracted t h e expanding and bread-making qualities of t h e flour are not affected.” The conclusions of Bremer,4 ‘in 1907, are also t o t h e effect t h a t t h e soluble proteins have little bearing on flour strength. Rousseaux and Sirot,‘ in 1913,consider t h e ratio of total nitrogen t o soluble nitrogen as a valuable index t o baking value and have determined a n ideal ratio for flours according t o their method, as well as t h e limits between which strong flours must fall in this respect. G E N E R A L CONSIDERATIONS-NUmerOUS other results of careful and valuable research might be cited, but t h e above serve t o indicate t h e confusion existing in t h e present state of our knowledge regarding t h e factoi-s involved in flour strength, a n d is intended t o serve this purpose rather t h a n constitute anything like ea complete summary of all the work which has been done in this field. Numerous summaries of this sort have been published in text-books and articles dealing with methods of milling and baking technology, such as t h a t of t h e Jagos,G and a repetition of them here would serve no useful purpose. It is believed, moreover, t h a t t h e above discussion indicates nearly all of t h e view-points from which t h e problem of t h e chemistry of flour strength has been attacked. The situation is very well expressed by Bailey? when he says: “Perhaps one of t h e reasons t h a t a greater degree of success has not attended these endeavors is t h e fact t h a t i t has been attempted t o discover one constituent (or group of constituents) which is t h e sole determining factor. It does not seem reasonable t o believe t h a t in so complex a substance as wheat flour t h e percentage of one constituent can be regarded as solely indicative of baking quality. Rather must we study these various compounds in their relation t o one another, in a n effort t o arrive a t their single a n d combined effects.” “The Quantitative Extqaction of Diastases from Plant Tissues,”
1
J. Am. Chem. Soc., 36 (1914). 759-770. “Studies on Wheat Flour. Influence of H-ion Concentration on Baking Value of Flour,” Compt. rend., 10 (1911), 170-206. a M i n n . E x $ . Sta. Bull., 54 (1897). 4 “Hat der gehalt des Weizemehles an Wasserloslrchen Stickstoff einer Einfluss aul seiner Backwert,” Zbschr. Unter. N a h r . Genuss.. 13 (1907), 2
69-74.
“Les matikes azote& solubles comme facteur d’appreciation des farines,” Compt. rend, Adad Sci., 156 (1913), 723-725. 6
cit. “Relation of the Composition of Flour t o Baking Quality,” Canadzan Mzller and Cevealist, 5 (1913), 208-209. 8
7
LOC.
Vol. 8. No.
2
P U R P O S E OF T H I S I N V E S T I G A T I O N
I n a series of investigations of the various factors which may inffuence t h e strength of wheat flour, now in progress in t h e Division of Agricultural Chemistry of the University of Minnesota, i t was proposed t o study the chemical constitution of t h e various proteins in flour with a view toward ascertaining more definitely t h a n has yet been done, whether or not t h e proteins of a strong flour may differ in their chemical constitution from those of a weak flour, since t h e physical properties of their glutens are found t o differ so markedly. Wood,’ in 1907, following Osborne and Harris’ modification of Hausmann’s method, subjected samples of gliadin and crude gluten (composed chiefly oE gliadin and glutenin) of flours of different strength, t o hydrolysis for 8 hours with strong hydrochloric acid. He then steam-distilled the products of hydrolysis with magnesia and determined t h e percentage of nitrogen given off as ammonia. Finding a close agreement in t h e different samples he concluded t h a t gliadin and glutenin of all wheat flours are of t h e same chemical composition. Since t h e work of Wood,’ a more detailed method of protein analysis, ‘which gives further insight into t h e constitution of t h e protein molecule and is capable of yielding quantitative results, has been presented by Van Slyke,2 who has incidentally shown t h a t t h e hydrolysis of gliadin with strong hydrochloric acid is not complete at t h e end of 8 hours. It was therefore decided t o make further study of the chemical constitution of flour proteins in the light of better methods of analysis now available. METHODS
OF
STUDYING
CHEMICAL
COMPOSITION
OF
PROTEIXS
It has been shown repeatedly t h a t for practical considerations all of t h e nitrogen of flours of the higher milling grades may be regarded as in t h e proteins. The chemical structure of t h e proteins has been clearly demonstrated by Fischer3 and a host of other workers since, so t h a t i t needs no elaborate discussion here. Briefly stated, t h e facts appear t o be t h a t the protein molecule is made up of a number of amino acids, there being some 1 8 or 20 of these which occur in natural proteins. These are probably linked together by anhydride combinations between t h e amino group of one amino acid a n d t h e carboxyl group of another. This is indicated by the nature of t h e products formed (amino acids) when the protein is subjected to hydrolysis, Moreover, it appears t h a t t h e characteristic chemical a n d physical nature of individual proteins depends largely on t h e nature and number of t h e various amino acids of which they are composed. I n a comparison of t h e chemical constitution of proteins, then, i t is necessary t o split t h e molecule by hydrolysis into its “bausteine” (characteristic units) and determine t h e relative proportions of these which are formed in each case. There is no known method of ascertaining LOC.cit. “The -4nalysis of Proteins by the Determination of the Chemical Groups Characteristic of the Different Amino Acids,” J . Biol. Chem., 10 (1911). 15-55. 8 “Untersuchungen iiber Aminosiiuren. Polypeptide und Proteine,” Berlin, 1899-1906. 1
2
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THE JOURNAL OF INDUSTRIAL A N D ENGINEERING CHEMISTRY
the exact manner in which these units are grouped together in t h e various proteins, since even t h e sensitive anaphylaxis reaction is not specific in t h e case of many vegetable proteins, as has been demonstrated by Wells and Osborne,’ who found t h a t animals sensitized with gliadin of either wheat or rye will react with hordein of barley, a protein known t o have a different chemical constitution, a n d t h a t gliadin a n d glutenin, known t o be different as regards t h e relative proportions of the various amino acids in their molecule, react anaphylactically with each other. METHOD USED I N DETERXINING PRODUCTS O F PROTEIN HYDROLYSIS
Van Slylte’s method gives t h e most detailed insight into t h e protein molecule of any known method which, a t t h e same time, indicates quantitatively t h e distribution of its component units. Accordingly, t h e Van Slyke method, in some cases slightly modified, was used in this investigation. T h e method, which is a n extension of t h e principle of t h e Hausmann method, consists of a division of t h e protein molecule into various groups, or units, after prolonged hydrolysis with hydrochloric acid, and t h e determination of t h e percentage of nitrogen in each individual group, thus ascertaining t h e distribution of t h e total nitrogen in t h e protein. Briefly, t h e groups determined are: ( I ) ammonia or amide nitrogen, which is considered TABLE I-DISTRIBUTIOX OF NITROGEN
I N THE
work. Their sources a n d relative baking values, as measured b y loaf volume, are indicated in Table I. T H E P R O D U C T S O F P R O T E I N HYDROLYSIS F R O M E N T I R E FLOUR
Osborne’ a n d his associates have shown t h a t there are five proteins present in flour, zliz., gliadin, glutenin, albumin, globulin a n d proteose, t h e latter being of little significance. The first two named compose t h e gluten, already referred to, while t h e others are soluble in dilute salt solutions a n d are, for t h e most part, removed in t h e familiar process of “washing out” t h e gluten. Since t h e proteins are, for all practical considerations, t h e only nitrogen compounds in t h e higher grade flours, i t was decided t o submit first, in all cases, a sample of the entire flour t o prolonged hydrolysis with strong hydrochloric acid, a n d determine t h e distribution of nitrogen in the various units. Should t h e results vary in different flours, i t would be necessary t o obtain t h e different proteins and ascertain their composition in a similar manner. If they should show t h e same chemical constitution then they must be present in t h e flour in varying amounts t o account for t h e difference when analyzed collectively, as is done in t h e hydrolysis of t h e entire flour. T h a t t h e latter is true has, of course, been concluded by numerous investigators who have extracted flour proteins with specific PRODUCTS OF HYDROLYSIS OF
BAKING
Sample NO.
B401 B438 B439 B440 B441 B444 B445 B452
FLOUR Pillsbury Patent P a t e n t Biscuit Flour P a t e n t Flour P a t e n t Flour High Gluten, Low Strength Flour Very High Gluten Flour ,“Fortyfold” Soft White Wheat Patent Flour
SOURCE
ENTIREFLOURS
STRENGTH
Loaf volume Northern Spring Wheat Good Poor Soft Missouri Wheat Good Xebraska Turkey Wheat Medium Hard Wheat, Prosser, Washinaton Poor North Dakota Wheat Medium Kansas Experiment Station Very Poor Troy, Idaho Poor Ritzville, Washington
t o be derived from -CONH2 or -CONHOCgroups linked t o t h e carboxyl groups of t h e dicarboxylic acids in t h e protein molecule (glutamic and aspartic acids) ; ( 2 ) humin nitrogen, .from t h e dark-colored pigment and slight amount of insoluble matter always formed in t h e hydrolytic products of acid hydrolysis of proteins; (3) t h e amino nitrogen of t h e mono-amino acids, which corresponds t o all of t h e mono-amino acids excepting proline a n d oxy-proline; (4) t h e nonamino nitrogen of t h e mono-amino acids, which corresponds t o t h e proline and oxy-proline; and ( 5 ) t o (8) t h e nitrogen corresponding t o each of t h e individual di-amino acids, i. e., arginine, lysine, histidine, and cystine, respectively. Thus, eight units of t h e protein molecule may be estimated quantitatively, t h e determination of histidine nitrogen a n d lysine nitrogen being subject t o a larger experimental error t h a n t h e other units, which may be determined with t h e exactness required by ordinary quantitative procedure.
141
TOTAL PER CENT O F TOTAL NITROGEN N Per cent 2,035 1.67 1.928 2.170 2.13 2.55 1.260 1.917
Ammonia Humin Basic N N N 20.81 5.32 8.10 18.85 5.62 9.25 21.01 4.92 8.56 21.47 5.00 7.88 21.03 5.16 8.74 23.00 5.42 7.08 18.21 7.51 9.00 19.87 5.63 8.03
Mo,noamino acid N 65.77 65.65 65.51 65.65 65.07 64.50 65.28 66.47
Eight flours of t h e higher grades (as separated in t h e process of milling) from various sources a n d of varying baking qualities were selected for t h e preliminary
solvents a n d have found their amounts t o vary widely in different flours. The solvents most frequently used are: (I) alcohol-varying from 50 t o 80 per cent, a n d ( 2 ) neutral salt solutions of different concentrations. The former was a t first thought t o extract only gliadin while t h e latter was considered t o remove only albumin, globulin and proteose. Owing t o t h e fact t h a t solutions of varying strengths and different methods of extraction have been employed by different investigators, however, their results often disagree widely, a n d in many cases even fail t o support t h e same general conclusions. Furthermore, i t has been found t h a t t h e solvents mentioned above are not as specific as was formerly supposed, a n d t h a t alcohol extracts not only gliadin b u t also considerable of t h e “soluble proteins,” t h e material so extracted depending on t h e strength of t h e alcohol, while salt solutions extract some gliadin as well as albumin and globulin, according t o t h e concentration of t h e solution. Other physico-chemical factors undoubtedly enter as well. Olson2 states t h a t “the amount of gliadin extracted by I per cent sodium chloride solution approximately amounts t o about 29 per cent of t h e total proteids,” and “the nitrogen
“Is the Specificity of the Anaphylaxis Reaction Dependent on the Chemical Constitution of t h e Proteins or on Their Biological Relations? The Biological Reactions of t h e Vegetable Proteins. 11,” Jour. Infect. Dis., 12 (1913), 341-358.
1 “The Vegetable Proteins” (1912). Plimmer’s, Monograph, London, New York, etc. 2 “Quantitative Estimation of Salt-Soluble Proteins in Wheat Flour,” THISJOURNAL,6 (1914), 212.
F L O U R S USED I N T H E INVESTIGATION
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T H E J O C R J A L OF I N D U S T R I A L A N D EiYGINEERING C H E M I S T R Y
bodies soluble in salt solution are partly or wholly soluble in diluted alcohols varying with t h e concentration of sodium chloride used.” T h a t a study of t h e products of hydrolysis of the flour proteins both collectively a n d individually can furnish a n indication of the proportions of these proteins in t h e flour, providing there is no difference in t h e chemical constitution of t h e same proteins in different flours, is evident from t h e following considerations: the percentage of ammonia nitrogen yielded on t h e hydrolysis of t h e individual proteins of wheat flour varies as follows, according t o Osborne, gliadin 2 4 . 5 , glutenin 18.8, leucosin (albumin of flour) 6.8, and globulin, 7 . 7 . Since the figures for t h e ammonia nitrogen show wider variation t h a n do those of any other units, and since also t h e estimation of this unit is probably accompanied by less error than that Of any Of the Others, it may be supposed that its estimation in the proteins taken collectivel,y and indicate closely the relative amounts of t h e various proteins present, providing, as mentioned before, t h e same proteins of different flours do not vary in their chemical constitution. In determining t h e distribution of nitrogen in t h e entire flour, ‘‘-gram samples were for 48 hours, and the ‘LHauSmann” units determined* In the Of the entire flour the presence Of a large amount of starch occasions a voluminous precipitate of “humin” material which made it impractical t o a t t e m p t a determination of all the units of t h e Van Slyke method, since a large enough not t o insure the estimation Of the units be with sufficient accuracy that the ‘Pres be of much significance. The instructions Of Van ”yke regarding t h e conditions for precipitating and washing t h e bases, however, were carefully followed. nitrogen in the I n determining the mixture, t h e presence of large amounts of “humin” substances resulting from t h e carbohydrates, and small amounts of fat, necessitated t h e slight modification of The \‘an Slyke’s method suggested by above mentioned substances make it impossible t o obtain a n aliquot until after they have been removed in t h e processes of determining t h e ammonia and humin nitrogen. Consequently, the mixture is evaporated ifl W K U O t o remove most of the hydrochloric acid. The ammonia is distilled off as in t h e Van Slyke process (mithout removing t h e materia1 from t h e distillation flask f r o m which t h e acid was evaporated o f f ) , collected in standard acid, and estimated b y titration; t h e humin filtered: Jvashed, and submitted t o Kjeldahl analysis for nitrogen, and total nitrogen determined in aliquot portions of t h e filtrate f r o m t h e humin. This, added to the ammonia nitrogen and t h e humin nitrogen, gives t h e total nitrogen in t h e hydrolyzed sample. S O correction \vas made in any of -the analyses for t h e solubilities of t h e bases in t h e solutions from which they Were precipitated, since t h e same conditions mere observed in all CaSeS a n d t h e results are strictly comparable. 1 “Studies on t h e Chemistry of Embryonic Growth. I. Certain Changes in t h e Nitrogen Ratios of Developing T r o u t Eggs,” J . Am. Chem. SOL.,36 (1913), 632-645.
Yol. 8, SO.2
The results given in Table I were obtained from t h e analyses of the eight samples of flour by the abovedescribed process. The different flours vary significantly with respect t o the ammonia nitrogen yielded on hydrolysis. T h e basic nitrogen or nitrogen of t h e diamino acids also shows a slight variation, this being inversely as t h e variation in ammonia nitrogen. The variations shown in the table are much greater than could possibly be due t o experimental error and were confirmed b y repeated determinations. Hence, there can be no doubt t h a t these variations show actual characteristic differences in t h e nitrogen distribution in t h e different samples, T H E DISTRIBUTIOS
O F S I T R O G E N I?: GLIADIN,
GLCTENIN,
AKD SOLUBLE PROTEIKS
Hydrolysis of the entire flour having shown characteristic differences in t h e composition of t h e entire protein material contained in them, i t appeared t o be necessary t o establish as definitely as possible R7hether or not the chemical constitution of the various individual proteins is the Same in different flours. For this purpose two flours which differed widely in their origin, total nitrogen content, and baking strength were selected. Flour B401 is a typical iliinnesota patent flour, milled f r o m northern spring wheat, of fairly high nitrogen content and of baking strength, while B438 is a patent biscuit flour, made f r o m a softer Missouri a,heat, low in total nitrogen and of poor baking strength. Gliadin was extracted from the gluten of the flours with alcohol and carefully purified b y pouring t h e concentrated syrup from the clear alcoholic extract alternately into large volumes of water a n d strong alcohol a n d finally digesting with absolute alcohol and ether, according t o t h e method of Osborne, Glutenin was also prepared according to Osborne7s method which consists, briefly, of dissolving the residue left after t h e alcohol extraction of the crude gluten in a dilute solution of potassium hydroxide, neutralizing m,ith hydrochloric acid t o precipitate the glutenin, decanting the liquid and further extracting the precipitate repeatedly with alcohol t o remO\ye t h e remaining gliadin; finally digesting with absolute altohol and ether. The preparations of glutenin in this work were not pure, being contaminated by small quantities of carbohydrates, oxving t o lack of facilities for obtaining clear extracts and filtrates at t h e time, but it is believed that all other nitrogen-contaking bodies !$-ere removed, and t h a t t h e preparations served the purpose of the investigation, namely, to ascertain whether there was any appreciable difference in t h e chemical constitution of t h e pure proteins. Considerable quantities of each of the tlvo flours were then extracted TT7ith I per cent salt solution, the extracts were filtered as clear as possible and concentrated ilz L~acuo. ~ h e s eextracts and weighed quantities of the gliadin and glutenin \?rere then hydrolyzed for 48 hours with strong H c ~ . T h e gliadin and glutenin were analyzed according to t h e Van Slyke method, while only ammonia nitrogen was determined in t h e case of t h e soluble proteins. From the results shown in Table I1 ( I , 2 , and 3) it is readily seen t h a t , after making allowance €or the limits of experimental error
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T H E JOURNAL OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY
of the method, there is no apparent difference in t h e chemical constitution of t h e proteins of typical strong a n d weak flours of t h e same market grade. THE
OF
DISTRIBIjTIOP;
XITROGEK
IN
CRUDE
GLUTEN
More complete evidence t h a t t h e gluten-forming proteins are of t h e same chemical constitution in differe n t flours was obtained by analyzing thoroughly washed crude glutens of three flours o f widely differing characteristics. he Same twoflours as in t h e immediately preceding experiments were used, and in addition, B444, a Kansas flour of exceedingly high nitrogen a n d gluten content,but of low baking strength, as shown in Table I. The results, obtained from t h e complete Van Slyke process as applied t o t h e crude glutens from these three flours, appear in Table I1 (4) and indicate
tion, then t h e humins combine with t h e ammonia a n d thereby become nitrogenous.” I n order t o ascertain whether varying proportions of starch would influence t h e results obtained b y t h e Van Slyke method as used in these investigations, a sample of t h e flour B401, t o which had previously been added 2 0 per cent of its weight of wheat starch, was hydrolyzed a n d t h e distribution of nitrogen in t h e products of hydrolysis determined. There was no significant change in t h e Percentage of ammonia nitrogen when compared with t h e sample t o which no starch was added, although there was a very noticeable increase in humin ?i a n d a corresponding decrease in basic N, as shown in Table II1* Further, it has recently been shown b y Gortner a n d
S I T R O G E R I N THE PROTEINS OF TYPICAL STRONG (1)-In t h e Gliadin (2)-1n t h e Glutenin Van Slyke’s Method Van Slyke’s Method N PER CENTOF TOTALN PER CENTOF TOTAL B401 B438 B401 B438 (Strong) (Weak) (Strong) (Weak) UNITSDETERMIXED 25.90 16.50 16.17 Ammonia N . , , , , , , , , , . , , , . . , , . . , , , , . , , , , , , . 26.13 .................... 0.50 0.57 1.84 1.66 0.18 Cystine AT.. . . . . . . . . . . . . . . . . . . . 0.29 0.18 .............. 4.47 9.69 9.27 .......................... 6.77 5.62 5.47 7.59 0.97 Lysine N . . . . . . . . . . . . . . . . 2.61 1.90 I n Filtrate from Bases: 53.59 53.38 Amino N . . . . . . . . . . . . . . . . . . . . . . . . . 53.46 54.10 Son-amino N . . . . . . . . . . . . . . . . . . . . 7.44 7.55 9.52 9.35
TABLE11-DISTRIBUTIOX O F
TOTAL ................................. (3)-Ammonia
OF
THE
SOLUBLE
PER CENTOF TOTAL N B401 B438 B444 (Strong) (Weak) (Weak) 22.87 23.19 23.69 1.19 1.37 1.11 0.46 0.70 0.43 5.24 5.54 5.54 2.79 1.28 1.50 2.21 2.60 2.28 55.21
56.15
__
__
9.54 -
9.88 -
10.13 -
99.97
99.47
99.40
99.50
99.51
100.71
99.82
B401 12.68
B438 12.84
PROTEIKS
IN
AFFECTING T H E NITROGEN DISTRIBUTION I N FLOUR
I t is evident t h a t t h e differences in t h e percentages of ammonia nitrogen and basic nitrogen yielded on t h e hydrolysis of t h e several entire flours, as shown in Table I , cannot be accounted for as being due t o differences in chemical composition of t h e individual proteins since i t has been clearly shown t h a t these have t h e same chemical constitution. It was thought a t first t h a t t h e varying percentages of starch in t h e different flours might cause differences in t h e percentage of ammonia nitrogen, since Mann,’ in 1906, states (‘if in addition t o t h e carbohydrate, ammonia or other nitrogenous substances are in solu“Chemistry of the Proteids,” New York, 1906.
WEAE FLOURS (4)-In Crude Glutens
__
h-itrogen Yielded on Hydrolysis of the Soluble Protein.
SIGNIFICAKCE
AXD
__
t h a t not only are t h e gluten-forming proteins in flours of widely differing baking qualities of t h e same chemical constitution, b u t t h e rati.0 of gliadin t o glutenin is probably t h e same, or very nearly so, in flours of t h e same market grade b u t very different baking strengths. With respect t o this latter point, i t may be said t h a t since, as is shown above, gliadin yields 26 per cent of its nitrogen as ammonia nitrogen after hydrolysis, while glutenin yields only 16 per cent of its nitrogen in this fraction, t h e determination of ammonia nitrogen of t h e hydrolyzed glutens will certainly indicate a n y significant variation in t h e ratio of gliadin t o glutenin in different flours, although the limits of experimental error are not narrow enough t o indicate very small variations in this ratio. The d a t a in Table I1 (4) indicate very clearly, therefore, t h a t there is no significant variation in t h e gliadin-glutenin ratio in flours of such widely varying baking strength as those used in this investigation. THE
I43
......
55.14
Blish’ t h a t other carbohydrates t h a n starch which might be present in flour do not affect t h e percentage of ammonia nitrogen obtained after hydrolysis, although they may, in many instances, increase the humin nitrogen by forming condensation products of huminlike nature with tryptophane. The significant variation in t h e percentages of ammonia nitrogen in t h e different flours (Table I) must therefore be due t o considerable variations in t h e amounts of soluble proteins present, since t h e gliadinglutenin ratio does not differ enough t o account for TABLE 111-EFFECTOF STARCH ON PRODUCTS
OF
THE DISTRIBUTIOK OF NITROGEN IN THE HYDROLYSIS O F FLOUR B401 Per cent of the total nitrogen
Ammonia Humin MATERIAL USED B401 . . . . . . . . . . . . . . . . . . . . B401 2 g. Starch . . . . . . . .
+
N
N
20.81 21.05
5.32 6.38
Basic
N
8.10 6.95
Mono-amino acid 1; 65.77 65.62
t h e difference in t h e percentages of ammonia nitrogen. As already shown, t h e “soluble proteins” (albumin a n d globulin) yield respectively 6.8 per cent a n d 7.7 per cent of ammonia nitrogen on hydrolysis, while t h e gluten yields about 2 3 per cent. I t may be calculated, therefore, t h a t flours containing t h e larger amounts of “soluble proteins” will yield t h e smaller percentages of their total nitrogen as ammonia nitrogen after hydrolysis. Accordingly, t h e flours showing t h e lower ammonia nitrogen figures as shown in Table I might be supposed t o contain t h e larger percentages of their proteins in t h e form of albumin and globulin. Since there is no known method of quantitatively estimating t h e albumin and globulin in flour, owing t o t h e fact t h a t t h e extraction of t h e various proteins 1 “On the Origin of the Humin Formed by the Acid Hydrolysis of Proteins,” J. Am. Chem. Soc., ST (1915).
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varies with the concentration of t h e solvents employed, t h e proportions of solvent t o material extracted, and possibly other physico-chemical .factors, t h e determination of ammonia nitrogen in hydrolyzed flour, flour extracts, and gluten should form a basis for a more exact knowledge of the proportions in which t h e various proteins occur in flours. Such methods are obviously unadapted t o ordinary analytical use, but afford t h e best possible method of exact study and careful investigational work. For example, in Table I1 ( 3 ) , the per cent of ammonia nitrogen yielded on hydrolysis of t h e extract with I per cent salt solution indicates clearly t h a t protein other t h a n albumin and globulin was extracted, since, as already pointed out, t h e per cent of ammonia nitrogen yielded by pure albumin and globulin should be lower. It is suggested also t h a t t o ascertain how much albumin and globulin are extracted by alcohol of any given percentage i t should suffice t o hydrolyze t h e alcoholic extract and determine t h e percentage of ammonia nitrogen in a similar manner. Since this article has been in press, t h e results of a study of t h e purity of proteins extracted from flour by various solvents, using methods here indicated, have been published by Bailey and Blish.’ I n order t o substantiate t h e evidence t h a t there is a relation between t h e ammonia nitrogen yielded on hydrolysis of flour, and t h e total quantity of soluble proteins in t h e flours in question, t h e latter were extracted with t a p water (since it was thought desirable t o use t h e same solvent as was used in washing out t h e glutens) a n d t h e percentage of “soluble nitrogen” in t h e total nitrogen of t h e flour estimated in the extract. Although protein material other t h a n globulin and albumin is extracted in this process, t h e results should be comparative, a n d in t h e order as indicated above, t h a t is t o say, t h e previously indicated relationship between ammonia nitrogen of the hydrolyzed entire flour a n d t h e soluble nitrogen should be apparent. T h a t this is true is indicated by t h e results obtained, as shown in Table IV. TABLEIV-COMPARISON O F PERCENTAGES O F SOLUBLE NITROGEI I N ENTIRE FLOUR N I T B AMMONIA NITROGEN AFTER HYDROLYSIS OF ENTIREFLOUR Per cent of total N TOTAL Sample No. NITROGEN Soluble N Ammonia N B444 ..................... B440 ..................... B439 ..................... B401 ..................... B452 ..................... B441 B438 ..................... B445 .....................
.....................
2.55 2.17 1.928 2.085 1.917 2.130 1.67 1.26
15.68 18.20 18.67 18.94 19.55 20.42 26.86 28.96
23.00 21.47 21.01 20.81 19.87 21.03 18.85 18.21
Inspection of Table IV shows t h a t , with t h e single exception of B441, as the percentage of ammonia nitrogen increases, t h e percentage of soluble nitrogen decreases, as was expected from t h e theoretical considerations already discussed. CONCLUSIONS
I-The individual proteins of strong a n d weak flours are identical in their chemical constitution, as determined by Van Slyke’s method for t h e analysis of proteins. [Since this went to press, t h e attention of the writer 1 “Concerning t h e Identity of t h e Proteins Extracted from Wheat Flour b y t h e Usual Solvents,” J . B i d . Chem., 28 (1915). 345-357.
1’01. 8, S o .
2
was called t o a study of some of the physical constants of gliadin from flours of varying strengths, by Gr6h a n d Friedl,’ in which they conclude t h a t proteins of different flours have t h e same constitution.] 11-The ratio of gliadin t o glutenin is much more nearly constant in flours of different baking qualities t h a n has heretofore been supposed. 111-There is a far greater variation in t h e percentages of t h e so-called “soluble proteins” (albumin and globulin) in flours. IV-Since t h e various proteins in t h e same flour differ widely in their content of ammonia nitrogen, t h e determination of ammonia nitrogen in flours, in extracts of flours made with various solvents, and in t h e crude gluten of flours, after their previous complete hydrolysis with strong mineral acid, can be made t o serve as a n accurate indication of t h e amounts of t h e various proteins present, since t h e proteins of widely different flours have been shown t o have t h e same chemical constitution. Acknowledgment and sincere thanks are herewith extended t o Professor R. W. Thatcher under whose supervision this work was done, also t o Dr. R. A. Gortner and Professor C. H. Bailey for many valuable and helpful suggestions. DEPARTMENT O F AGRICULTURE UNIVERSITYOF MINIESOTA ST. PAUL, MINXESOTA
THE ANALYSIS OF MAPLE PRODUCTS, V Miscellaneous Observations on Maple Syrup Incidental to a Search for New Methods of Detecting Adulteration2 B y J. F. SNELL Received August 20, 1915
I n t h e course of a search for new and improved methods of detecting adulteration in maple syrup, t h e author and his associates have made a number of miscellaneous observations which may be suggestive or otherwise useful to other investigators in this line. I-EFFECTS
OF SOME REAGENTS
It is well known t h a t lead subacetate produces a heavy precipitate in maple syrup and t h e normal acetate one not so heavy. I n t h e “malic acid value” determination, alcohol and calcium chloride are employed together as precipitants. I t appeared to the author not improbable t h a t a systematic search would reveal other precipitating reagents whose behavior might prove equally useful in the examination of maple products. Mr. J. M. Scott made some desultory experiments in this direction and has recorded in his notes “indifferent” results with ether, zinc acetate, uranium acetate, cadmium chloride, bismuth nitrate, copper nitrate and cupric chloride. Mercuric acetate gave a flocculent precipitate which settled on standing and was light yellow in color. Silver nitrate in 50 per cent solution gave a white precipitate which darkened on standing. The author has also observed t h a t barium salts give no precipitate in maple syrup although they do so in solutions of the Biochem. Zlschr., 66 (1914), 154. Presented a t t h e 51st Meeting of t h e Amencan Chemical Society. Seattle, Aug. 31-Sept. 3, 1915. Previous papers of this series-Tars JOURNAL,6 (1913), 740, 993; 6 (1914), 216, 301. 1
2
