INDUSTRIAL A N D ENGINEERING, CHEMISTRY
An intemting item in Thomacrset’s patent on the use of nickel (38) is the statement of the requirement of low oil absorption of the blue for maximum alkali stability. This would be indicative of a decreased total surface for the pigment particles, which would be accompanied by decreased powers of adsorption. To conclude, t h e constitution of the iron blueg is still in doubt. ,Most of the early work waa carried on and interpreted from a Jingle viewpoint only. Because of the complexity of the systems involved, simultaneous attacks on the problem from different angles are required, such as have been initiated recently by Weiser and others, Aging studies under suitable conditione, accompanied by chemical, colloidal, x-ray work;etc., would aid in resolving the controversies over this subject. The nickel and alkali stability problems are tied u p with the general problem of constitution, and further eluaidation of the latter will aid in explaining the facts assoaiated with the former. ACmOWLEDGM ENT
The suggestions, advice, and criticism received from David
Daddson and Joseph Greenspan, of the Department of Chemistry, Brooklyn College, are gratefully acknowledged. LITERATURE CITED (1) Abegg, R. W. € Handbuch I., der anorg. Chemie, Band 4, Abt. 3, Teil2B, p. 561. Leipzig, 9. Hirael, 1935. (2) Bever, A. K,van, Rec. traw. Aim., 57, 1269 (1938). (3) Bhattacharya, A. I., J. Zndion Chsm. Soo., 12, 143 (1935). {4) Zbdd., 18, 81 (1941). (5) Ibid., 18, 86 (1941). (6) Bhattacharya, A. K., and Dhar, N. R. Z., 2. amrg. allgem. Chem., 283, 240 (1933). (7) Brown, T. P. (to Interchemical Corp.), U. 8.Patent 2,269,516 (Jan. 13, 1942). Cambi, L., and Clerici, A., Gam. chim. iW.,58, 61 (1928). Chretien, P., Compt. rend., 137, 191 (1903). Coleby, L. J. M., A m . Sd.,4,207 (1939). Davidson, D., J. Chem. Education, 14,277 (1937). Davidson, D., and Welo, L. A., J. Phys. Chem., 32,1191 (1928). Eibner, A., and Gerstacker, L., Chm.-Ztg., 37, 137 (1913).
801
(14) Emeleus, H. J., and Anderson, J. S., “Modern Aspects of Inorganic Chemistry”, p. 139, London, G. Routledge & Sons. 1938. (15) Harrison, A. W. O., “Manufacture of Lakes dt Precipitated Pigments”. Chap. IX, London, Leonard Hill,1930. (16) Haael, F., and Sonun, C. H.,J . Am. Chem.Soc., 52,1337 (1930). (17) Hofmann, K. A., Arnoldi, H., and Heindlmaier, H.. Ann., 352, 54 (1907). K. A., Heine, O., and Hochtlen, F., Zbid., 337, 1 (18) Holm-, (1904). (19) Hofmann, K,A,, and Resensoheck, F., Zbid.. 340, 267 (1906). (20) Zbid., 342,364 (1906). (21) Keggin, J. F., and Miles, F. D., N&ora, 137, 577 (1936). (22) Levi, Q. R.. Oiorn. d i m . dnd. o & W , 7,410 (1926). (23) Mattiello, J., “Protective and Decorative Coatings”, Vol. 11, p. 263, New York, John Wiley & Sons, 1942. (24) Muller, E., J. prak Chsm., 84,353 (19lQ. (25) M., 90, 116 (1914). (26) Muller, E., and Lauterbach, H., Zbid., 104, 241 (1922). (27) Muller, E., and Stsnniach, T., Zbid., 79,81 (1909). (28) ZW., 80, 163 (1909). (29) Muller, E., and Treadwell, W.. Zbid., 80. 170 (1909). (30) Rabinerson, A., Kdloid-Z., 39, 112 (1926). (31) W i e n , H., and Zimmerman, W., Ann., 451, 75 (1927). (32) Rigamonti, R., Gam. chim. W.,68,.803 (1938). (33) Rollier, ha. A., and Arreghini, E., Ibid., 69,499 (1939). (34) Shack, I., and Wilson, E. A. (to Interchemical Corp.), U. 8. Patent 2,342,429 (Feb. 23,1944). (36) Skraup, H., Ann., 186, 371 (1877). (36) Thomssset, P. A. (to Anebaoher-Siegle Corp.), U. S. Patents 2,275,929 (March 10, 1942); 2,329,3&4 (Oct. 14, 1943). (37) U. S. Dept. of Commerce. Census of Manufactures, 1939. Colon, and Pigments,p. 2 (1948). (38) Van Wirt, A. C., and Jones, G. F. (tq Imperial Paper & Color Corp.), U.S. Patent. 2,357,296 (Sept. 5, 1944). (39) Weber, H. B., “Collmdd Salts”, p. 249, New York, McGrawHill Book Go., 1928. (40) Weiser, H. B., “Inorganic Colloid Chemiatry”, Vol. 111, pp. 307, 343, New York, John Wiley & Sons, 1938. (41) Weiser, H. B., Millimn, W. O., and Bates, J. B., J. Phys. Chem.. 45, 701 (1941). (42) Zbid., 46, 99 (1942). (43) Wilson, E. A., and Schack, I. (to Interchemical Corp.), U. 8. Patent 2,288,309 (June 30, 1942). (44) Woringer, P., Chsm.-Ztg., 36, 78 (1912). (46)Worinmr, P., J. prald. Chem., 89,51 (1912). (46) Wyrouboff, M., Ann. ddm. phya., [5] 8, 444 (1876).
PEANUT PROTEIN HYDRATES Preparation and Properties R. S. BURNE’l” S o u t h e r n Regional Research Laboratory U. S. D e p a r t m e n t of Agriculture, New Orlaans,
c
Y E R T A I N peanut and soybean protein-water interrelations have been observed which not only have significant theoretical aspects but, more important, open new fields of applic& cion for isolated peanut, soybean, and other vegetable proteins. The purpose of this paper is to describe some of these proteinwater relations and, in subsequent communications, to discuss their practical applications. The water relations of peanut protein have been successfully applied in developing new adhesives for gumming purposes, and in making flexible glues for the setup paper box manufacturer and the bookbinder. Protein-water relations involving hydration (bound water), awelliig, and related phenomens: are complicated and, in some respects, controversial. Lloyd (I2 ) states that “whatever may be felt to be the limits of ‘bound water’ in protein, it is generally agreed that water is held with varying degrees of force”. Astbury (I) says: “The water that proteins can take up seems to fall roughly into two kinds, the loosely and tightly bound.’’ It
La.
is also generally agreed that tightly bound water is bound to protein hydrophilic groups by hydrogen bonds, and t h a t it is possible to estimate the quantity of such bound water from a knowledge ofthe amino acid content of a protein (14). Concfrning the mechanism of the second type of water binding, Phillips (IS) points out that the polar groups of the protein “may not only associate with water molecules by coordination, but may also cause other wbter molecules to orient themselves and provide an outer sphere of less firmly bound water molecules”. Additional references and excellent summaries relative to more recent concepts of protein-water relations are given by Bull (S), Greenberg (IO), and Compton (7). The homogeneous, translucent, hydrated, peanut protein sole are disintegrated by the presence of urbound water, as indicated by the appearance of a second phase; the phenomenon is somewhat analogous to phase reversal of emulsions. This is apparent if th- protein hydrates are considered tu solutions of water in
862
VoL 35, No. 9
INDUSTRIAL AND E N G I N E E R I N G CHEMISTRY
protein. Addition of wster which the protein can no longer 6s- by a centrifuge equipped with a solid basket, and the centrifuged dispersion was clarified by filtration. The protein was then pred v e (or bind) remits in reversion to a auspenmon of protein cipitsted from the elnrified filtrate at 25’ C. by adding sulfurous (moreor less hydrated) in water. Although st pH values in or wid in an amount rsquired to attain the pH value shown in near the isoelectric range the protein suspension settles rapidly, Table I. The protein curds obtained were allowed t o settle relatively stable suspensions result when the pH is raised. overnight and were then separsted from the supematant liquor Greenberg (9)points oat that there are few criteria hy which and dried at 50” C. All three proteins shown in Table I were the hydrstion of proteins may be defined, and Bull (4) states that prepared under relatively mild conditions; that is, they were “bound water is d e h e d in terms of the experimental technique extraoted at nearly neutral pH vsluea to avoid contwt with SIby which it is measured”. In ths present paper all the water kdi, Bnd the opportunity for beat denaturation during drywhich a peanut protein will ahsorb at pH values between 4.5 and ing was minimieed. 9.0, without disintegration oi tht sol, to produce a second phase. The -e mild conditions mu& be employed to isolate soybeaa irs considered to be bound to the protein moleeules. Such one protein if it is to be auitnble for preparing neutral sol hydrates. phase systems w e called “hydrstes”. The exact manner by All oommercisl saybean proteins examined by the author form which water is retained hy the protein micelle may be debatable, gel8 at the high concentration requirsd to produca a hydrate of the but that point is not important for present purposes. type under consideratioo. This behavior ia probably due to Such views are not offered as supporting evidence for or sgaiust greater alteration in the structure or cornpasition of the protein m y particular theory of protein behavior. They are presented in during ita maoufsetun, than occum under mild extraotion and order to doscribe vsrious phenomena which occur with solutions drying conditions. of peanut protein; to diRerentiste solutions of water in protein (hydrates) from dispersions of protein (more or less hydrated) in water which cao bo prepared in the pH range 4.5 to 9.0; and to EYDRATES W m I N OR NFAR ISOELECTRIC RANGE. atimulste interest in t,hewsterrelationshipsof vegetable globulins. Peanut protein haa &o isoelectric reage between pH 4.0 and The poanut protein used in this investigation was a mixture 5.0 (X), and the same amount of protein o m be rewvcrod by uf se~eralincompletely characterimd proteins in undetermined precipitation with sulfur dioxide at nnv DH within this ranee ., 16). .. proportions. For tho prosent purposes the mixtures are r e f e d When peanut protein curds, to as peanut protein, and the prepared by precipitation at conditions under which each pH 4.5 (method 1, Table I), produet WLU prepared will be are dried at 50” C., a point given. .lithough soybean proPeanut protein hydrates ere deeeribed which =re eapsbls is reaehed st which tho intein forms similar hydratea, of binding in-sing amounts of water, fmm 30 to about the prexnt paper is limited terior of shell-dried lumps 01 70% by weight of the sol, a s the pH d u e of the system is I O peanut protein. curd becomes B tmnslwent, inoreased fmm 4.5 to 9.0. T h e pieaence of water in BXtacky sal whioh coolv to B of that bound by the pmteib renults in disintegration of PREPARATION OF PROTEINS viscous plastic mass. Results the protein-water %yetem. The protein hydrates are of numerons moisture detertacky and, at pH values near neutrality, have Viscosity The peanut proteins were minationsmade on Lhisplastic ohnraeteristies which make them suitable for use a s prapared from s o l v e n t m a t e d indicate that the adhesives, provided the pmtein used is isohted from the extracted peanut meal. To meal with a minimum of alterstion by heat or alkali. wster content is 38 1.0% cxtra.cr, the probin from the Precipitated and filtered peanut protein curds ara daat the point where the curds mcnl, one part of meal was watered by warming until the curd particles coalesce and &st fuse to form s sol. m i d with ten partsof dilute exdude unbound water. This reduction of the watsr When mster is added to d i u m hydroxide mlution at content from 7 0 8 0 to about 40W greatly reducas drying dried peaout protein I to raisa 25” C. and st the pH values costs in preparing isolated peanut protein. Similar the find mixture to approxinhown in Table I. The exean be prepared fmm isolated soybean protein. hydrates mately 38% water oontent. crwt was separated from the the dry protein quiokly abinsoluble fraction oi the meal
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September, 1945
INDUSTRIAL A N D ENGINEERING CHEMISTRY
the m a i l amount of water added. After about 30 minutes the slightly swollen protein particles can be kneaded with the fingem into s homogeneous msss that is translusecnt when p r d into a thin film. Tlie ma9s is not taeky at mom temperature, but on warming it becomes extremely tacky, m d has charaeteristios identical with those exhibited by the fused curds just described. When water is added to dry (fi to 8% MT~S
L.8. a U d pnrtidtr io r s U 2
Figure 2.
1
a
7.6 7.5 7.0
4.6 6.0 6.0
Gitratioo. The erne= water which -sins is then eliminated by ~ curd particles rarming and kneading the ounls. This C B U the to coalesce and exclude water which is present in exes4 of thst required to form the protein hydrate. The curds obtained by precipitation at pH 5.0 diRer from those obtained at pH 4.5 in that the former can be reduced readily by heating at 40-50" C. from a wnter content of 50 to 70% to one
S-U
mUd p.ctiota in d l 4
Phutumicrugraphn of Coagulated Pmteine ( X 250)
moisture) protein 1, in excess of that required to raise the find mixture to 38% water content, the protein psrticles become opaque after a short time and exhibit little tendenoy to ooalesce. This maximum water content of 38% at which B homogeneous, coherent, system oan be obtained is close to the 35% water reported to be bound to (isoelectric) proteins (e, 11). If B portion of the hydrated (38% water) protein with B pH value in the isoelectric range is sllowed to stand in exwater, tho surface of the hydrate becomes opsque, the suhstsnoe loses its cohesiveness, and the [lystem disintegrates. The disintegrstion proceeds rapidly if the hydrated protein mass is broken up by 8 glass rod. Van Der Dussen and Maaskant (16) state: "One speaks oi losa of oohesion when protein gels or similar systems disintagmte." In contrast to protein 1 (Table I), protein 2, which as precipitated from solution at pH 5.0 and then dried, yields a plastio sol that is tacky at room temperature when the wster oaittent is brought to 38%. The hydrate prepared from this protein, which has a pH at the upper end of the isoelectric range, will bind additional water up to about 42% before the system breaks down. Protein hydrates o m also be prepared directly from preoipitatted protein curds by removing part of the exwater by
2
863
80,%a8 801 gaq W a ~ r o a t d mth . 80, added $lowly
corresponding to the bound water cspsoity of the protein. The tiltered ourds coalesce as they are w-ed, exclude unbound water on kneading, and finally become an extremely coherent N ~ B B which can be pulled like taffy. even at room temperatures. The bound water remaining with the protein amounts to about 42.5%. Filtered protein curds precipitsted at pH 4.5 can be dew&& only with difficulty by WSrmiLIg and kneading. The forces within the individual hydrated particles appear to be too strong to permit easy coalescencewith surrounditib partides in the prcsenoe of, m d to the exclusion of, unbound water. The dewatered curd is "short" et room temperstwe and breaks when pulled. I t can he dewatered, however, to a mater omtent of 38%. This dewatering of the curds e m reduce drying cost8 in the manufacture of isolated protein, as will be emphasized in another papor of this series. A fair yieid of protein can also be obtained by precipitation st pH 6.0 (method 3, Table 1). When the protein dispersion is acidified by the slow Bddition of a saturated aqueous soiution of sulfur dioxide, thc pratein-ourd particlea, which separste by gravity om sisnding, eodesce at room temperature; a sol is pioduced instead of il precipitate of small psfticles of jelly (curds), similar to that formed by precipitation st pH 4.5 and 5.0 (methods 1 and 2, Tahie I). The viscous sol obtained after the supernetknt liquor is decanted has a water content of ahout 56%. It is not transiucent m d appesre to be 8 protein hydrate which is contaminated with finely divided curds. Although the protein fraotions precipitated s t pH 4.5, 5.0, and 6.0 may difier with respect to relative contents of the several protein component+ it is probable that the marked differences in physical properties of the dewatered protein curds (bydrstea) am due primarily to the pH of t.he curds and not to differences in
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INDUSTRIAL AND ENGINEERING CHEMISTRY
protein composition. These dewatered curds have physical characteristics identical with those exhibited by sols which are prepared from dried protein precipitated at p H 4.5 by adding water, or dilute alkali and water, to obtain the equivalent p H and water content of the dewatered curds. In each case the consistency of the hydrate decreases as the p H and water content increase. Further evidence that the p H of the curds exerts a greater influence on the physical properties of the dewatered curds than do the relative contents of protein components is indicated by the following experiment: A protein fraction prepared by precipitation at p H 6.0 and subsequently dried can be redissolved at pH 8.0 and reprecipitated at p H 4.5. The protein obtained has the same physical characteristics as prot,ein precipitated directly from a peanut meal extract at p H 4.5.
methods 1, 2, and 3 C M be dupersed to give a sol or gel, irrespective of the amount of water present. There is no justification for considering these approximations as more than qualitative evidence of the presence of unbound water. More important is the ability of the protein to bind more water as the pH increases. This makes it possible to prepare sole from the proteins in Table I that have sufficiently low viscosities between p H values 6.0 and 9,0 to permit their practical use as adhesives. Furthermore, the sols are tacky if excess water is absent,’as indicated by the presence of only one phase. I n the le= viscous hydraes, tackiness becomes apparent where a thin film of sol is applied to the surface to be glued. At p H values above 9.0 the tackiness of peanut protein sols becomes too slight to be of practical value, and the more concentrated mixtures undergn gelation;
EFFECT OF ADDED ALKALI ON HYDRATE SYSTEMS
The effect of alkali on the hydrate system was determined by the addition of varying amounts of water and dilute sodium hydroxide to dry protein 1. It was found that as the p R of the system is raised, increasing amounts of water can be added to the homo-“ geneous, translucent, hydrate system before disintegration takes place. Other changes accompany this increased hydration. I n the isoelectric range the cohesiveness, viscosity, and tackiness of the hydrates are at a maximum, and the amount of bound water is at a minimum. As the p H of the hydrate is raised to 6.0, additional amounts of water can be added before disintegration occurs, and less force is required to disintegrate the hydrates, which indicates a decrease in cohesivenbss. The force considered responsible for the disintegration of the protein micelles at p H values above the isoelectric point is the osmotic pressure which is brought about by an excess of diffusible ion resulting from the Donnan distribution. Although a gelatin gel is elastic and equilibrium is eventually established on swelling, the micelles of the peanut protein sols are not elastic, and the cohesive forces are so weak that the protein micelles are easily ruptured. The effect of p H on the swelling of peanut protein is difficult to evaluate by the techniques employed for gelatin, because at pH values above the isoelectric range the water-soaked protein particles disintegrate. At p H values of 6.0 and below, the coagulated protein particles are large enough to settle out in the excess water, and although they appear to consist of white amorphous particles, microacopic observation reveals that they consist of transluscent hydrated jellylike particles. At p H values between p H 6.5 and 9.0, the coaghlum obtained upon the addition of excess water is a fine colloidal suspension, which becomes increasingly finer as the p H increases. At p H values above 9.0 the addition of water does not produce a second phase. Figure 1 is a photograph of peanut protein hydrates prepared at pH 5.0 and 6.85, together with the effect of excess water on the system. Photomicrographs of the coagulated proteins (Figure 2) show the effect of p H on particle size. The point at which the hydrates disintegrate is not sharp, especially a t the higher p H values. I n general, within the p H range 4.5 to 6.0 the hydrates can be brought to a water content of 38 to 50% before the system disintegrates; within the p H range 6.0 to 7.0 the limit is 50 to 60%; and between p H 7.0 and 9.0 the limit is 60 to 70%. At p H 9.0 and above, proteins prepared by
Vol. 37, No. 9
INFLUENCE O F SALTS
Obviously, salts influence the properties of colloidal system* of the type described. When isoelectric peanut proteins are leached with excess water t o remove salts and other nonprotein material, the consistency of the protein-water systems increases materially. In all unleached protein preparations, the products contained a t least part of the natural salts of the meal, as well as P portion of the salts formed during the preparation of the protein and remaining in the dewatered curds. Therefore, duplicate batches of both dewatered curds and dried proteins from dewatered curds, prepared at a given pH value, will cbntain approximately the same salts, generally in uniform minimum amounts. The effect of salts on the viscosity of peanut proteiri solutions prepared at lower concentrations and higher p H value* has already been discussed (6). ACKNOWLEDGMENT
The author wishes t o express his appreciation to M. E. Jefferson, Mary L. Rolliis, and C. H. Billett for the photographs. LITERATURE CITED
(1) Astbury, W.
Soo., 36. 871-80 (1940). (2) Bull, H.B., “Physical Biochemistry”, p. 233, New York, Johu Wiley & Sons, 1943. (3) Ibid., Chaps. XI1 and XVIII. (4) Ibid., p. 235. (5) Burnett, R. S.,and Fontaine, T. D., IND. ENG.CHFX., 36,284-8 (1944). (6) Burnett, R. S.,Roberts, E. J., and Parker, E.D., Ibid., 37,27681 (1945). (7) Compton, E. D., J. Am. Leather C h m . Assoc., 39,74-90 (1944). ( 8 ) Fontaine, T. D., and Burnett, R. S., IND.ENQ.CHEM.,36,164-7 (1944). (9) Greenberg, D. M.,in Schmidt’s “Chemistry of Amino Acids and Proteins!’, 2nd ed., p. 481, Springfield. Ill,, C. C. Thomas. 1944. (10) Ibid., Section VII,pp. 1139-44. (11) Ibid., p. 1139. (13) Lloyd, D. J., and Shore, Agnes, “Chemistry of Proteins”, 2nd ed., p. 476, London, J. A. Churchill Ltd., 1938. (13) Phillips, Henry, J. Intern. Soc. Leather Trades Chem., 18,165-74 (1934). (14) Sponsler, 0.L.,Bath, J. D.,. and Ellis, J. W., J . Phya. Chem., 44, 996-1006 (1940). (15) Van Der Dussen, A. A., and Maaskant, L., in Schmidt’s “Chemistry of Amino Acids and Proteins”, 2nd ed., p. 450, Springfield, Ill, C.C. Thomas, 1944.
T.,Trans. Faraday
