----
Lime Hvdrolvsis of Soybean Protein J
J
FOAMING PROPERTIES OF THE HYDROLYZATE JOSEPH M. PERRI, JR.', AND FRED HAZEL 3
Department of Chemistry and Chemical Engineering, University of Pennsylvania, Philadelphia, Pa.
E
YDROLYZATES can be prepared from Alpha soybean pro: tein which foam strongly and are suitable for combating fires. Since solutions of amino acids do not have the property of forming durable foams, complete hydrolysis of the protein is both unnecessary and undesirable. Lime has been used (8)in the reaction, and with this reagent the hydrolysis is incomplete unless excessive time is employed. The purpose of the present investigation was to study the effect of varying the protein-lime ratio and the duration of the hydrolysis on (1) the efficiency of lime in peptizing or otherwise solubilizing the protein, and (2) the extent of the hydrolysis as indicated by the nature of the constituents and the alpha-amino nitrogen content of each of the hydrolysates. It was desired, also, to identify the foaming constituents in the hydrolyzates.
T h e hydrolysis of Alpha soybean protein with calcium hydroxide has been studied under varying conditions. The effects of the variation of the protein-lime charge and the duration of hydrolysis on the composition of the hydrolyzate have been determined. The extent of hydrolysis under varying conditions is evaluated from the alpha-amino nitrogen content of a complete l i q e hydrolyzate. It is shown that excess lime increases the extent of hydrolysis, and it is postulated that this effect is due to the surface of the solid lime. The active foaming agents of a solution of partially degraded Alpha protein have been isolated and identified. The effect of the duration of lime hydrolysis on foaming properties of hydrolyzate has been determined.
metaprotein by adding trichloroacetic acid until its concentration in the system reached a value of 16%. Maximum precipitation occurred under these conditions, and no interfering hydrokysis was produced. This procedure was used by Siebert (7). The proteose fraction was obtained by saturation of the metaprotein-free solution with zinc sulfate (1). This treatment precipitated both protein and proteose, but since the protein fraction was known, the proteose fractiop could be calculated, The peptone and hexone base fraction was obtained from the filtrate of the proteose precipitation by adding a 5% solution of phosphotungstic acid in 5% sulfuric acid. The remaining amino acids and lighter pe tides were estimated on the basis of the nitrogen content of the J t r a t e from the phosphotungstic acid precipitation. Table I shows the effectsof varying the protein-lime charge and the time of hydrolysis on the percentage composition of the filtrates. Data are included for the a-amino nitrogen contents of the filtrates and for the percentage hydrolysis calculated by using 75.7% as the value of the completely hydrolyzed protein. The effect of variation in conditions on the percentage yields and on the amount of organic matter in the residues are shown in Tables I1 and 111, respectively. Table IV compares the organic matter content of the sludges, calculated on the basis of no hydrolysis, with the experimental values.
HYDROLYSIS O F PROTEIN
The protein used in the experiments was obtained from the Glidden Company, and is known commercially as Alpha protein. It had a nitrogen content of 14.91%. Baker's calcium hydroxide, U.S.P., was employed in the degradation. Hydrolysis was conducted by mixing a weighed amount uf lime with 1000 cc. of water in a 2-liter, three-necked, round-bottom flask and heating to boiling on an oil bath. A known weight of protein was added and the system heated, with stirring, at 95-98' C. for a stated time. The mixture was filtered, and the filtrate and solid residue were analyzed separately. Some nitrogen was lost as ammonia during the hydrolysis and as nitrogenous material in the residue. The percentage yield of protein matter in the filtrate was found by analysis for total nitrogen and comparison with the amount of the element present in the original protein charge. Determination of ash and organic matter in the residue gave the magnitude of the loss caused by failure of the lime t o react with the protein. The percentage hydrolysis was found by comparison of the aamino nitrogen contents of the filtrate and of the completely hydrolyzed protein. a-Amino nitrogen determinations were made by the Van Slyke method (9). The filtrate was analyzed for soluble degradation products by first precipitating the various fractions and then determining the nitrogen in the successive filtrates by the Kjeldahl method (8.
DISCUSSION OF LIME HYDROLYSIS
Van Slyke (9) proposed, as a criterion for following the extent of degradation of proteins, the measurement of the a-amino nitrogen content of the hydrolyzate. H e &owed that the aamino nitrogen content increases with time and reaches a limiting value which corresponds to the completion of hydrolysis. Figure 1 presents the effect of time on the extent of hydrolysis of Alpha soybean protein with lime, as measured by the a-amino nitrogen content of the hydrolyzate. The break in the curve was caused by the fact that, after 198 hours of hydrolysis, the hydrolyzate was removed from contact with organic matter in the sludge and the filtrate was digested further with lime. The lime added to further the hydrolysis presented a fresh surface which may have preferentially adsorbed the remaining higher fractions. This would decrease the amount of nonamino nitrogen and result in an increased percentage of a-amino nitrogen in the hydrolyzate, Upon complete hydrolysis, 75.7% of the nitrogen content of the hydrolyzate was in the a-amino form, Table I C shows that, starting with Alpha soybean protein, the hydrolysis process consists of degrading the protein into derived bodies of varying complexity which yield amino acids as the final product. The derived protein fractions, in order of complexity, are metaprotein, protein, proteoses, and peptones.
ANALYSIS.Metaprotein was obtained by adjusting the pH of the solution with hydrochloric acid to 3.4. This corresponded to the hydrogen-ion concentration a t which maximum precipitation occurred. The method for determining the p H of maximum precipitation was described by Loeb (6). The protein fraction was obtained from the filtrate of the Present addresa, National Foam System, Inc., Philadelphia, Pa.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
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The data obtained bv varvinr: " - the orieinnl Drotein and lime charges ?Table I, A and R) are for Amino runs of 10 hours. Because of the short duration ----ChargeAleraProProPepAcid 6 a-r\mino Protein, Lime, Time, protein, tein. teose, tone, Peptine, Sitro- Hyclrolyof the hydrolysis compared to the time required for grams grams hours C, cz % 7c '; gen, % ms, yo complete hydrolysis (more than 300 hours), t,he reA . \-ariation of Protein Charge actions n-ere far from complete. As a result the 125 45 10 5 .2 225.9 8.3 116.3 0.6 31.2 24.5 19.0 25.1 filtrates contained high percentages of derived pro80 45 10 3.8 30.2 23.5 23.5 31.0 40 45 10 3.1 lL5 21.8 31.8 27.8 28.4 37.5 teins and comparatively lox percentages of amino acids. The data show that, as the relative amount B . Variation of Lime Charge of lime was increased, there mas a decrease in the 25.9 16.3 80 45 10 3.8 30.2 23.8 23.5 30 31 o0 10 3.5 23.6 16.7 32.7 23.5 23.1 80 35 higher-molecular-weight fractions and an increase 10 4 9 23.8 11.5 35.1 24.7 22.3 29.5 80 25 27.3 20.0 25.3 20.3 17.1 80 15 10 7.1 22 5 in the lower fractions. The same effects were pro30.9 14.6 23.2 13 2 80 10 10 18 6 11.7 10.0 duced by increasing thc time of hydrolysis 'C. Variation of Time of Hydrolysis (Table I C). 30.7 45 5 9.7 26.9 13.7 19.0 18.2 80 24.0 An increase i n lime charge had the same effect as 25.9 10 30.2 23.5 31.0 3.8 16.3 23.8 45 80 0.8 31.3 24 41.4 an increase in the time of hydrolysis, but for a dif45 80 Trace 0 :3 9:9 zi:s 6,s:2 55.5 73.4 98 45 80 ferent reason. The fact that the pH values for all 0.0 6.6 198 0.0 17.6 15.8 63.6 84.0 45 80 .. .. 71.4 .. 250 80 45 systems containing 25 grams of lime or more were 94.3 .. .. 75.65 100.0 307 80 45 .. 75.7 .. 360 the same and remained the same (within 0.2 100.0 45 80 pH unit) throughout the 10-hour hydrolysis period, demonstrated that not all of the lime charge \?as required to maintain the system in a Table I C indicates that there n-as a rapid decrease in the metasaturated condition. The effect of larger quantities of lime in 'promoting hydrolysis must be attributed to the effect of the solid protein and protein fractions with duration of hydrolysis until a t surface, introduced as solid calcium hydroxide, on the reaction. 98 hours the metaprotein fraction was too small for quantitative A complete explanation for the increased reactivity is not availestimation. After 198 hours had elapsed, both the metaprotein and the protein, the more complex of the fractions, were shown to able. However, it is knonm that both native and derived probe absent (after 307 hours the proteose fraction was absent). teins are adsorbed strongly at interfaces. On this basis it seems Thus, it is apparent that the amounts of higher fractions present plausible to assume that the increased hydrolysis encountered in the hydrolyzate also yield information concerning the extent under these conditions was promoted by adsorption of one of the reactants on the solid surface of the other. of hydrolysis. The preceding discussion does not imply that the course of the The effect of excess lime in promoting the conversion of Alpha hydrolysis need follow the pattern: original protein -+ protein soybean protein into soluble hydrolytic fractions is also revealed ---+ proteose +peptone +amino acid. It means only that by the data of Table 111. There was a decrease in the percentage the simpler fractions are formed by degradation of the more comof organic matter in the sludge as the n-eight of lime in the charge was increased. Since this same trend in percentage composition plex, The break in the a-amino nitrogen-time curve is of interest in this connection. The break represented a change in the rate of the sludge would have occurred even if there had been no hydrolysis (the ratio of protein to lime being decreased). it is of a-amino nitrogen formation to total nitrogen in the system, and necessary t o compare the experimental results obtained in the was caused by the removal of the hydrolyzate from the sludge which contained organic matter. The only fractions present in cases of hydrolysis with those calculated for no hydrolysis. This comparison is made in Table IT' and Figure 2, where the proteinthe hydrolyzate a t the time of the separation were the proteose, peptone, and amino acid fractions, If unreacted protein was n lime ratio is plotted against the percentage organic matter in the sludge. Curve I represents the values calculated from the proconstituent of the sludge, it may be postulated that it was the direct precursor of the lighter fractions. It remains a possibility, tein-lime charge. The prytein-dispersing capacity of the lime solution (10 grams for 1000 cc. of sahrated solution) was taken however, that metaprotein and protein were being formed, but into consideration in making the calculations. It was assumed that the rate of degradation of these heavier fractions compared that this weight of protein would be dispersed in every case, and to their rate of formation wa? so high that the amounts prcwnt the rest would go into the sludge. Fere below the limits of qualitative detection. Curve I1 is constructed from the value of percentage organic matter in the sludge from the 80-25 gram run. This value wm selected because 25 grams of lime was the apparent minimum necessary t o maintain the system in a saturated condition (i.e., constant pH) during hydrolysis. It w-as assumed that this system contained no excess lime2 and that the percentage organic matter in the sludge represented the amount to be expected in
TABLE I.
EFFECT O F v.\RI.\BI.l hydrolyzntc and con-
a a
-
LITERATURE CITED
TABLE T'I.
1SFI.C.J;XCE
iX Content,
ST.IB1LITY
System
PH
Gram/Liter
Stability Value, Seconds
I I1 I11
7.6 7.6 8.9 8.5
0.1098 0.1098 0.1098 0.1098
2040 1380 3120 5160
IV
TABLE VII. System A B
C
a 15%
tions.
O F IIETAPROTEIN O S F O A M
I N F L U E S C E O F LIETAPROTEIX OK 4PhCITY
c
PH
S Content, Gram/Liter
Cc. Liquid Converted t o Foam
11.5 11.6 11.35
0 . 0344Q 0.0344 0.0229
57 73 110
FOAMING Foaming C a aoity, C C . ~of. Pi 11,060 14,160 32.020
of these amounts were employed in the foaming capacity determina-
( I ) Alien, "Cornmfrrial Organic ,innly;is", 5th eii., Vol. 8, p . 710 Philadelphia, Biakiston C o . . 1930. ( 2 ) .lssoc. of Official Agr. Chem., Official arid T e n t a t i v Afethods of Analysis, 5 t h e d . , p. 26, p a r a g r a p h 2 2 (19.101. (3) C l a r k a n d Ross, ISD. ENG.CHEM.,32, 1594 (1940). (4) Gray a n d S t o n e , FVaZZersteinLab. Commun., 3, KO.10 ,15!1 ( 1 '340). ( 5 ) Loeb, "Proteins a n d t h e T h e o r y of Colloidal Behavior", 211d ed., p. 192, N e w York, 1\IcGram-Hiil Book C o . , 1924. (6) S h o r t e r , Phil. M a g . , [6] 27, 718 (1914). (7) S i e b e r t , J . B i d . Chem., 70, 264 (1926). (8) U r q u h a r t , E. S.P a t e n t 2,269,958 ( J a n . 13, 19.12;. (9) V a n Slyke, J . Bibl. Chem., 9, 194 (1911); 1 2 , 276 (19121; 16, 121 (1913); 23, 407 (1915). BASED on a dissertation presented by Joseph M. Perri, .Jr., t o thP Graduate School, University of Pennsylvania, in partial fulfillment of the requirements for the degree of dogtor of philosophy.
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