A STUDY OF THE NON-PROTEIN NITROGEN OF WHEAT FLOUR.*

13~ M. J. BLISH.

(From the Chemistry Department of the Montana Agricultural Experiment Station, Bozeman.)

(Received for publication, December 26, 1917.)

Preliminary to a projected investigation concerning the more important biochemical changes which occur when wheat is frosted, atd well as the relation of these changes to bread-making value, and particularly in order to study the effect of premature freezing on the nitrogen compounds of the wheat kernel, it has been found desirable to develop a more satisfactory m&hod for the separation of protein from non-protein nitrogen compounds in wheat flour than an examination of the literature has revealed. A number of methods have been developed for the separation of protein from non-protein nitrogen in almost all kinds of bio- logical products, and several of these appear to be satisfactory for their purposes. With cereals, however, none of these methods seems to answer the purpose since cereals contain alcohol-soluble proteins, which are not encountered in any other plant or animal tissues.

Reagents ordinarily used for precipitating proteins, such as alcohol, acetic acid, trichloroacetic acid, salts of heavy metals, colloidal iron, aluminum hydroxide cream, phosphotungstic acid, and tannic acid, are for various reasons unsatisfactory for re- moving gliadin from water extracts of flour.

Ritthausen,l 40 years ago, advocated the quantitative removal of the prot,eins from milk, by alternately adding to the protein in solution dilute copper sulfate and potassium hydroxide until the proportions were such that, the copper precipitate would no longer redissolve. The insoluble copper-protein compounds were then

*Published with the approval of the Director of the Montana Agri- cultural Experiment St,ation.

l Ritthausen, H., J. prakl. Chem., 1872, v, 215.

551

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552 Non-Protein Nitrogen of Wheat Flour

removed by filtration. This principle in modified forms has since been made use of by different investigators, although the accuracy of the separation of protein from non-protein nitrogen by this method has always been questioned.

Osborne and Leavenworth2 have recently reported a study of copper-protein compounds, using edestin and gliadin in their ex- periments, and have found that if the correct amount of copper sulfate is added to a solution of gliadin in dilute sodium hydroxide solution, the gliadin and copper are both practically completely precipitated. As they state, however, there are several points to be definitely cleared up if this procedure is to be made a basis for the accurate separation of protein from non-protein nitrogen compounds in extracts from biological material.

When the principle of Osborne’s procedure was tried’ with flour extracts in this laboratory, it was found that more nitrogen was removed by this means than by the use of any other of the re- agents previously mentioned. Phosphotungstic acid removed nearly, but not quite, as much and tannic acid slightly less. More- over, the copper-protein precipitate filtered readily, giving a water-clear solution, which could easily be concentrated under reduced pressure to one-twentieth of its original volume. That there was probably no copper in the filtrate, other than that which was in combination with amino-acids and peptides, was indicated by the fact that no blue color was perceptible, nor were positive tests with potassium ferrocyanide obtained until after consider- able concentration. The method seemed so simple of manipula- tion that it was decided to test the effectiveness of the separation by ascertaining whether or not all protein was removed, as well as to test the copper-protein precipitate for free amino nitrogen as an indication of the presence or absence of less complex nitrogen compounds in the copper-protein precipitate.

In Table I are data which indicate the total nitrogen which is not precipitated from a water extract of a standard patent flour (Ceretana) by the copper method, as compared with the total nitrogen not removed from the same extract by tannic acid, col- loidal iron, and phosphotungstic acid, respectively. The figures represent averages of several determinations by each method.

2 Osborne, T. B., and Leavenworth, C. S., J. Biol. Chem., 191f5-17, xxviii, 109.

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M. J. Blish 553

TABLE I.

Method of treatment using 50 cc. portions of extract.

1. 1 cc. Merck’s 5 per cent colloida1 iron ppt. at room tem- perature, followed by 1 cc. of concentrated MgSOa solution................................................

2. Same as No. 1 with precipitation at boiling temperature.. 3. Precipitation with 10 cc. of 10 per cent phosphotungstic

acid after making strongly acid with HCl.. . . . _. . . . . . . 4. Precipitation with tannic acid.. . . . . . . . . . 5. Coppermethod..........................................

Total N not precipitated.

0.00266 0.0033

0.0010 0.0012 0.0008

The details of the copper method as used on flour extracts in this laboratory are presented in the following experiments.

Experiment I.

Ceretana, a standard patent flour milled by the Bozeman Milling Company of Bozeman, Montana, was used in the pre- liminary work, which consisted of determining the proper pro- portions of flour to water for the extraction, the length of time of extraction, and a comparison of the copper method with the other methods mentioned earlier in this paper with respect to the ‘(total non-protein nitrogen” determined by other methods. Distilled COz-free water, saturated with toluene, was used for all extractions. After trying various proportions of flour to water, 20 parts of water to 1 of flour was decided upon as a convenient proportion to use for extractions. Extractions were carried on for varying lengths of time, using portions of 20 gm. of flour to 400 cc. of water in 500 cc. Erlenmeyer flasks. The flasks were shaken vigorously every 15 minutes, for periods of 2, 3, 4, 5, 6, and 12 hours, respectively. At the end of each period the ex- tract was filtered through paper, the proteins were precipitated* by treating 50 cc. of extract with 15 cc. of 0.1 N NaOH, followed by 16 cc. of 0.1 N CuSO+ and total nitrogen was determined in the filtrate from the copper precipitation. The amount was found to be the same for the 2 hour extraction as for the 12 hour period, and therefore a minimum extraction period of 2 hours was adopted. Perhaps the most satisfactory method for the

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554 Non-Protein Xtrogen of Wheat Flour

dctc:rmiriation of “ fofnl non-prot.cin nitrogen” in flour extracts by the above rnct~hod is t,o pip&e 100 cc. of flour oxtract, into a 200 cc. sugar flask, add 25 cc. of 0.1 N NuOH followed by 27 cc. of 0.1 N CuS04, shake vigorously scvcrsl times until a iv:tter- clear supcrnatant liquid remains sftcr the precipitate settles, make to the mark, filter, and detrrminc nitrogen by the lijt~ltlahl rnrthod in 100 or 150 cc. of the filtrate.

Thc~ effectivtncss of the method NW then cxaminccl by study- ing the nature of the nitrogen compounds left’ in the filtrate from the: copper-protcin prceipitstion. In this work, t,wo flours, 11 and R, were used. Flour A was the Ccretana patent, flour men- tioned previously, while I), was a flour milled from some slighbly frost4 Karkov wheat crown in Montana. 1 liter port,ions of filtrre(1 flour ext,ract were placed in 2 liter flasks, and 400 cc. portions of 0.1 N NaOH were added, followed by 400 cc. of 0.1 N (:llli;(~~. Then small portions (of about 10 cc. conch) of 0.1 x (h801 wcrc atldcd, tlic whole Iwing ncll sl~akcn and allo~ved to stt.tlc sftcr each addition, until an absolutely clear, colorless supcrnatant, liquid remained after th(x precipitate settled. It uppenrs to be wOsolufcly taecessary thut the 0.1 x alkali he kept iv, pnruflimlined cot~faitwrs, or else be tnctde up fresh for each occasion, as it was found that alkali containing disiolvccl glass n-ould neithel cause a s~~arp separation nor give a clc:w, colorless filtrate. F’i- nally, tlist,illed wat,er was added up to the 2 liter mark, and the filt,ration pcrformecl .on a large paper filter. The filtration pro- ceeds rapidly and easily when the precipitation is made in the mnnnrr described. The filtrate was then slightly acidified with acetic wid and conccntratcd under diminished prcssurc to one- tncnticth of its rolunic. The concentrated solutions were slight,lJ viscous on acrourit~ of dissolved carbohydrate material, and gave a slight test for copper with ferrocynniclc~, which they did not give beforc concentration. l’hey also sh~wd the ,slightest t.racc of color due to copper salts. The concentrated solutions arc re- ferrrd t,o in the rernninder of this paper as Solut.ion g. These solutions gave no perceptible biuret reaction of any kind, but did give a very faint Millon’s reaction, and also a slight, Adamkicwicz reaction. The latter two reactions arc indicntive of the presence of the amino-acids, tyrosine snd tryptophane, but, do not ncces- sarily shon that t.hey a.rc in protclin combination. Solution S

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M. J. Blish 555

gave a slight color with Nessler’s reagent, indicating but a trace of free ammonia. Amino nitrogen was determined in Van Slyke’s micro-apparatus using 5 cc. portions of Solution X. Amide nitrogen was determined by diluting 50 cc. of Solution X to 100 cc., boiling for 2 hours with 2.5 cc. of concentrated sulfuric acid, according to the method of K6nig,3 and distilling with an excess of calcium hydroxide under reduced pressure. In order to find

.out approximately the nature of the rest of the nitrogen in the extracts, 25 cc. portions of Solution X were hydrolyzed with strong hydrochloric acid for about 12 hours, after which am- monia and amino nitrogen were again determined, and the in- crease in these constituents over the amounts as determined before hydrolysis was considered as indicative of the nature and complexity of any nitrogen compounds still present in peptide or protein form. In Table II are presented results of analyses of flours A and B.

TABLE II.

Constituent determined. Flour A (sound

patent).

pm cent

Total N of flour.. . . . . . . . . . . . . . . , . 1.89 Per cent of total N of flour extracted

by distiIIed COz-free water in 2$ hours............................... 16.06

Per cent of total flour N not precipi- tated by copper.. . . . . . . . . . . 1.62

Free ammonia in Solution X.. . . . . Trace. Amide N in total “non-protein” N.. . 20.00 Gm. of amide N in 100 gm. of flour.. a-Amino N in tota “non-protein” N.. 6.62 Gm. of amino N in 100 gm. of flour. . . Ammonia N after hydrolysis. . . . . . . 10.91 a-Amino N increase due’ to hydrolysis. 19.34 Humin N after hydrolysis.. . . . . . :. . . . . 8.16 Residual nitrogen by difference.. . . 34.97

T

I

-

Flour B ‘slightly ‘rested) ,

pm cent 1.79

15.40

2.12 Prace. 26.12

7.79

10.32 20.22

7.37 28.18

Flour A. Flour B.

pm.

0.006

0.002

rm.

0.0095

0.003

It will be observed from Table II that the actual quantity of ‘free amino nitrogen in a normal flour is exceedingly small, being about 2 mg. for every 100 gm. of flour. There is about three

SKijnig, J., Chemie der menschbchen Nahrungs- und Genussmittel, Berlin, 4th edition, 1910, iii, 274.

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556 Non-Protein Nitrogen of Wheat Flour

times as much nitrogen in the acid amide form. The figures for amino nitrogen vary widely from those obtained by Swanso’n and Tague4 who determined amino nitrogen in a patent flour using SBrensen’s form01 titration method and report about nine times as much as is found in these experiments. They used flour extracts without attempting to remove the proteins. Van Slyke micro determinations were made directly on some flour A extract in this laboratory in order to obtain a more satisfactory comparison with their method. The presence of the proteins made it impossible to take an adequate concentrated sample so t,hat determinations had to be made using 5 cc. of the extract, corresponding to 0.25 gm. of flour. This, according to the method used by Swanson and Tague, should give about 0.1 cc. of nitrogen gas which may be easily measured in the micro-appa- ratus. In no case, however, could more than 0.02 cc. be ob- t,ained, while the average of four determinations was about 0.01 cc., which is too small an amount to be considered for the pur- poses of a satisfactory determination, although quite in accord- ance with the findings using the Van Slyke method on the con- centrated extract from which the proteins had previously been precipitated with copper; i.e., Solution X.

Table II shows an increase in ammonia and amino nitrogen in Solution X after prolonged hydrolysis with strong acid. The increase, however, is not great enough to indicate the’presence of any considerable amount of protein. It suggests, however, that there is some nitrogen in the form of peptides which escapes pre- cipitation by the copper method, although it does not preclude the possibility that traces of protein ma,y still be present also. Hart and Bentley,5 studying the non-protein nitrogen of some feeding- stuffs, found Ohat after treatment with Stutzer’s reagent some nitrogen of the nature of peptide linkings remained in solution. The same seems to be true when Osborne’s procedure is followed, although it is probable that the precipitation of the true proteins is practically complete if the proper manipulation is followed.

4 Swanson, C. O., and Tague, E. L., J. Am. Chem. Sot., 1916, xxxviii, 1098.

6 Hart, E. B., and Bentley, W. H., J. Biol. Chem., 1915, xxii, 477.

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M. J. Blish 557

Experim.ent II.

In order to obtain a more satisfactory idea of the nature of the pcptidc linkings left in solution after t.he copper treatment, a portion of Solution ,X from flour A was acidified and treated with phosphotungst.ic acid. A considerable precipitat,e formed which contnincd 40.91 per cc‘nt, of the total nikogen in the solution not- withsbanding the fact, that when the original water extrwct of flours is treated with phosphotungstic acid no more (and often less) nitrogen is precipitatbcd than by t.he copper method. However, when Solut.ion X was subjtxked to a further treatment with cop- per, as in the first rcmovd of the proteins, no more nitrogen was brccipitated. Furthermore, when a portion of Solution X was misc4 with about nine times its volume of 95 per cent alcohol, 30 per cent of the t,ot,al nitrogen in the solut.ion was precipitated, along with the destxins 2nd other carbohydrates insoluble in alcol1ol. Tl~se facts strongly suggest the presence of peptide linkings of a less complex nature than true protein. The phos- photungstic acid precipitate was filtcrcd off, washed with a solu- tion of phosphotungstic and hydrochloric acids, and hydroIyzed for about 12 hours with st.rong hydrochloric acid, after which ammonia and amino nitrogen wcw determined. Amino nitro- gen was also determined in the filtrate from t,he phosphotungst.ic acid precipit,ation. Although the determinations were m& on small amounts of material, they wcrc confirmed I)y sc>vcral closely ngrceing duplicates and blanks on each analysis, t 50 result being reported in Tablr III.

TABLE III. ~-

Constituent determined in terms of total N in Solution X. Flour A.

N precipit.ated by alcohol.. _. . . . . S precipitated by further treatment with CuSOa and XaOH.! X precipitated with phosphotungstic acid. . . . . Total amino 9 in Solution S.. . . . . Total amino S in filtrate from phoxphot.rmgstic acid pre-

cipitatior~......,..............,.....,....,............... Amino S from exposed amino groups of pcptides (by dif-

ference).................................................. Ammonia S after hydrolysis of phqsphotungstic acid pre-

cipitate.................................................., ilmino N after hydrolysis.. . . . . . . , . .I

-I..

Per cent

30.00 None. 40.91

6.62

-1.81

I.531

5.71 14.30

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558 Son-Prokin Nitrogen of VCrhcatm Flour

*Is lIP:lI.ly 2s 1nq lx, nscc~rt:~incd from clc~tcitllill:lt.ions with Such sndl :mlounts of mat,c~ri:tl 3s arc nwcssit~tcttl in the prc- ceding ospcrimcwts, the fig:o~~s rcportccl in T:ll)le III sho\v that there cannot hnvc been much, if any, protein left in solution rift cr the first pwcipit:Ltion hy t.hc copper method. The amino tdrogcn tlctcl,lnill:~t,iolls on Solution S hcfore and after precipi- tation with phosphotungsiic acid sho\v t.hnt there is 1.81 per cent less nmino nitrogcw after the prccipitntion than heforc. This must8 lx CIIW to t,hc cxposc~l amino groups of the nitrogenous nlnttcr pwcipitxtcd l)y the phvsphotungstic ncitl, and nlthough thr tliffcrcncc involved the: mwsurcmwt of only 0.03 cc. of nitrogcw gas in the micro tlctc~~~iliIlatior1, it was confirmed 1)~ rcpc~itctl clctrrniirintioris which ngrwd prnctically as closely as the hrett~r could lx wad. 11ftc.r hyclrolysis there was about cbight, tiinw 3s much xriino riitrogcw, which clearly indicates t,hat tlw prccipitat.cx consiutcd chiefly of ptrptides of n less complex nature than protein. The amino nitrogc>n in the filtrate from tllr trc:tt.lrwnt, of Solut.ion S with phosphot.ungstic a&l which ~~nlountctl to -1.81 per crnt probably originntcd from the free ;mrino-ncitls in tlw cstIx:t. There is a much larger cliscrcpuncy I)etwcc?n the pcrcrntage of total nitrogen precipitntctl by phos- pl~otungstic wit1 :mtl tht sum of tjhe percent~nges of :nnmonia and :trniI<o nitrogen nft,cr hydrolysis khan nll~y r~:~~n:~l~ly IX a~-

rounted for by t,lie non-amino nitrogc~rl of the pc>ptitlc compounds. This stems t,o intlicnte that thcrc arc otllcr lxisic nitrogen corn- polln(ls in flour fstracts, whosct n:itur(: is :M yet lur.gc:ly ~~1lkI~O~VIl.

:I Itiurexicle kst, for purincs rcsu1t.d nclg:Lt ivdy. 11~1 :lttcml)t wks m& Do ascertain whct.her or not the copper

met hoc1 removes along with the proteins any nppreciable amounts of amino-xids or pcptide-like compounds of a nature less com- plcs than that of the proteins by redissolving some of the cop- per-protein prccipitak in gl:xid :wctic acid and tcst,ing t,hc solu- tion for free amino groups since the prescncc of consitlerahle copper dots not, intwferc: with the rc:~ction bctwcen the amino groups of amino-:lcitls and nitrous ncid. Accordingly :t solut.ion of the copper prtcipitat,c containing 4 mg. of nitrogen was intro- c~uccd into t.hc \7:~n Slyke lnicro-:tpp:lrnlus, bnt there was no

c~vitlence to show that any such compounds were present in the l)rcqipitate. Thcrcfore, it. is not lxdic~veci that, :my serious Crror

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M. J. Blish 559

from this source is introduced in the case of flour extracts, not- withstanding the fact that under certain conditions copper is known to be capable of forming insoluble compounds with a few amino-acids, though not with the majority of them.

When water’extracts of flour were allowed to stand for several weeks at room temperature in the presence of toluene, and ana- lyzed at intervals for non-protein nitrogen by the copper method, a gradual increase in non-protein nitrogen by autolysis occurred although there was no sign of putrefaction. The amount of total non-protein nitrogen doubled during the period from May 25 to July 6, 1917. This indicates that the method is appli- cable to proteolysis studies in flour.6

SUMMARY.

1. Practically a complete separation of protein from non- protein nitrogenous substances in water extracts of wheat flour may be accomplished by treating the extract with 0.1 N NaOH followed by 0.1 N CuS04 until there is but slightly more CuSO4 than an exactly equivalent amount of NaOH. The method is simple of manipulation and leaves no troublesome excess of the reagents employed for the precipitation. The method permits of rapid .filtration through ordinary filter paper, giving a water- clear solution which may be readily concentrated to one-twen- tieth its original volume, for determinations of amino nitrogen by Van Slyke’s micro method, and for amide nitrogen determinations. Some peptide nitrogen is not precipitated by the copper method, but the removal of the true proteins is practically complete.

2. Normal patent flour contains but about, 2 mg. of amino-acid nitrogen for every 100 gm. of flour, and about three times as much nitrogen in free acid amide form.

3. There is probably a considerable amount of non-protein ni- trogen not precipitated by the copper method which is neither amino-acid nitrogen nor is it in the form of peptide complexes; its nature is not known.

4. The method is applicable to studies of proteolysis or other studies involving the estimation of protein cleavage products in wheat flour.

5. It is not unlikely that the method will be found applicable to biological extracts from other sources than wheat and flour.

6 Acknowledgment is made to Miss Erma Lessel, who performed a large part of the preliminary analytical work.

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I? Csna~ 1. Lot 970. These records show that young rats are unable to grow on a mixture of equal parts of whole

wheat, maize, and rolled oats, together with butter fat to supplement the small and inadequate content of fat-soluble A in these seeds. In Period 2, the addition of 1.0 per cent of sodium chloride induced a slight acceleration of growth. That the cause of the slow growth rests in the inadequacy of the inorganic content of the ration, is made evident by the records of Lot 959, Chart 2.

Lot 971. These curves show that the addition of calcium alone (Period 2) to mixtures of wheat, maize, and rolled oats does not permit growth on this seed mixture. The seeds which we have studied are without exception too poor in cal- cium, sodium, and chlorine, and all must be added before growth can approximate normal and be long sustained (com- pare Lots 970 and 714, Chart 1, and 959, Chart 2, respectively). The protein content of this mixture of seeds is not of very good quality and the plane of protein intake of animals on this and the closely similar ration described is below the optimum but suffices to support normal growth over a considerable period (compare Lot 714).

Lot 714. These records illustrate that the addition of 3.7 per cent of a complete salt mixture, in Period 2 after 6 weeks of complete suspension of growth, enabled the animals to resume growth at nearly the normal rate on a diet of equal parts of wheat, maize, and rolled oats. The first limiting factor in this mixture is its inorganic content, and sodium, chlorine, and calcium are the only elements which need to be added. We have abundantly demonstrated that these three seeds are too poor in the fat-soluble A to support normal health over a prolonged period. The quality and amount of protein furnished by this seed mixture are below the optimum, but the effects of this would not become manifest except perhaps in failure of the animals to attain the full adult size, or in case the special burden of repro- duction is placed upon them.

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CHART 2. Lot 959. Even after practically complete suspension of growth during 7 weeks, on a mixture of wheat, maize, rolled oats, and butter fat, three of these animals were able to resume growth at once at a rapid rate, when both sodium chloride and calcium carbonate were added to the diet (compare Chart 1, Lots 970, 971, and 714). What is true of this mixture of seeds is true likewise of still more complex mixtures of seeds (compare Lot 715, Chart 6, and Lads 930 and 713, Chart 7).

Lot 714 B. It has been shown that the wheat, maize, and oat kernels are too poor in the fat-soluble il to maintain normal health in animals over long periods (3). Stunting and xero- phthalmia being the most prominent sequelae of this type of specific starvation (11). The ad- dition of a liberal amount of butter fat (fat-soluble A), after a period of stunting, exerts no influence upon the ability of the animals to grow. The first limiting factor is the inorganic eontent of the food mixture (compare Lots 970, 971, 714, Chart 1, and Lot 959, Chart 2, re- spectively).

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CHART 3. Lot 1,011. These records illustrate the fact that an improvement in the protein content of the diet by the inclusion of 10 per cent of casein, in a mixture of wheat, maize, and oats, does not enable the animals to grow. While it is a matter of importance in determining the vitality of the animals that the protein mixture in this ration be improved and that more of the fat-soluble A be added, the experiments here described emphasize the fundamental importance of having a satisfac- tory mineral content in the diet.

Lot 1,012. These records are of special interest when compared with the preceding charts. In Period 1, the mixture of wheat, maize, and oats was improved with respect to protein by the addition of casein, and fat-soluble A by the addition of butter fat. Although no growth was possible on this mixture because of the lack of sufficient calcium, sodium, and chlorine, growth was extremely rapid in Period 2 when these salts were added. This result is doubtless due in some measure to the mainte- nance of vitality in Period I of Lot 1,012, better than in Period 1 of Charts 1 and 2, in all of which experiments more than one dietary factor was operating at the same time to depress the vitality of the animals.

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169 I j, ,- , /i J /

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CHART 4. Lot 870. These records show the supplementary relations between the proteins of the wheat and oat kernels. The deficiencies of these seeds were otherwise made good. Under these conditions so low a plane of protein intake as 7 per cent sufficed to induce growth to about 80 per cent of the normal adult size and at somewhat less than the normal rate.

This resuIt is distinctIy better than was obtained with 7 per cent of protein derived in equal amount from maize and oats (Chart 5, Lot 869), or from maize and wheat (Chart 5, Lot 868). There is distinct improvement in the quality of these protein mixtures over the biological values of the proteins of each of the seeds when fed separat,ely (1). With rations of the latter type reproduction has never been observed. No young were reared on this ration because of the inability of the mothers to produce sufficient milk on proteins of this quality and in such limited amounts.

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CHART 5. Lot 869. This illustrates the value of the mixture of maize and oat proteins when fed at the plane of intake of 7 per cent of the food mixture. The deficiencies of the grains with respect to inorganic elements and fat-soluble A were made good by suitable additions. The results indicate that this protein mixture is inferior to that derived from the wheat and oat kernels (Chart 4, Lot 870), for growth was not so rapid, and the animals were somewhat more stunted than with the latter ration. The reproduction records on this ration are distinctly below those of Lot 870, on the wheat and oat mixture. Mixtures of maize and oat proteins in equal proportions do not appear to be greatly superior to the proteins of either seed fed singly at the same plane of intake, but some superiority is evident, since no reproduction has been secured on 9 per cent of either maize or oat proteins alone.

Lot 868 shows that wheat and maize proteins when fed together in equal proportions are not of so good a quality as are those of wheat and oats fed together in a similar manner (Chart 4, Lot 870). The fact that the one female produced three litters of young shows clearly that the mixed proteins are better than those of each of the seeds fed singly. These records together with those in Charts 4 and 5 show for mixtures of two cereal grains what we have previously demonstrated for mixtures of maize and navy beans, sir., that the biological values of mixtures of proteins from seeds are distinctly lower than that of the proteins of milk, and emphasize the fact that poor quality of the protein content of the diet is in all probability one of the factors in lowering the vitality of those peoples who live during the winter season on a diet restricted to a few arti- cles, the chief one being corn bread or wheat bread. Corn bread, salt pork, and molasses constitute in winter almost the en- tire source of nutriment of many people in the South today.

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160

Lao

60

40

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k CHART 6. Lot 715. These records illustrate the failure of young rats to grow when fed solely on a mixture of four

seeds, maize, wheat, rolled oats, and hemp seed. The prompt response with growth even after long stunting, upon the addition of a suitable salt mixture, adds further support to the belief that the inorganic content is the greatest factor in rendering mixtures of seeds incapable of supporting growth.

Lot 722 shows that growth cannot take place in the young rat on a monotonous food mixture consisting of maize 90 per cent and flaxseed meal 10 per cent. Even after almost complete suspension of growth during 3 to 4 months, there is an immediate response with increase in body weight on the addition of a suitable salt mixture to the diet. The flaxseed differs from the cereal grains in its relative richness in the fat-soluble A. This fact accounts at least in part for the great favor which this substance has found as a stock food.

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160

120

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CHART 7. Lots 930 A and 930 B illustrate the impossibility of obtaining growth on a restricted diet of maize, wheat, rolled oats, and millet seed in equal amounts. In Period 2, on the addition of salts, growth at once took place. a complete salt mixture, while Lot 930 B was given only sodium chloride and calcium carbonate.

Lot 930 A was fed

good in each case. The response was equaily

Lot 713 illustrates the failure of young rats to grow when given a monotonous ration of five seeds, and (Period 2) the fact that the limiting factor is the character and amount of the inorganic portion of the diet. Mixtures of seeds of which millet seed is a constituent will be adequate in their content of fat-soluble A provided not less than 20 to 25 per cent of millet seed is present. The diet of the female throughout the experiment was that of the male in Period 2.

Seeds of plants can be classed together without exception in their dietary properties, in that they must be combined with other foods which carry a much greater amount of calcium, sodium, and chlorine in order to render them complete from the dietary standpoint. In lesser degree the poor quality of the proteins of seeds and seed mixtures and the low cont,ent (with few exceptions) of the fat-soluble A are contributing factors in causing stuntin table foods.

g of animals fed too largely on this class of vege-

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M. J. BlishNITROGEN OF WHEAT FLOUR

A STUDY OF THE NON-PROTEIN

1918, 33:551-559.J. Biol. Chem. 

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