Peptization of Soybean Proteins Extraction of Nitrogenous Constituents from Oil-Free Meal Acids and B a s e s with and without Added Salts ALLAN K. SMITH AND SIDNEY J. CIRCLE U. S. Regional Soybean Industrial Products Laboratory, Urbana, 111.1
T
HE recent rapid increase in the production of soybeans
in this country has made available to industry a cheap domestic protein in practically unlimited quantity. The present production of protein is used mostly in the preparation of cold water paints and of sizes for paper and textiles. However, research indicates a much broader field of application in the form of adhesives, leather finishes, paper coatings, and plastics. The meal residue obtained from the solvent extraction of soybean oil contains approximately 45 per cent protein, and with the development of industrial uses for this product, the problem of efficiently separating and purifying the soybean protein grows in importance. A search of the literature does not reveal sufficient data for a rational approach to this problem or for a critical analysis of the processes now in use. Horvath's recent publication (8) on the chemistry of soybean protein extraction, although interesting, presents no additional experimental work. The most extended work in this field is that of Satow (IC),who investigated the separation of soybean protein and studied its properties and possible industrial applications. The procedures, as revealed by the patent literature (1,d, 3, 9),differ little from those described by Satow. The protein is extracted from oil-free meal by dilute alkali or alkaline salts, followed by precipitation with acid. Since the optimum conditions for this procedure are not recorded, it seemed desirable to study quantitatively the dispersion of nitrogenous constituents of oil-free soybean meal over a wide range of hydrogen-ion concentrations, using suitable acids and bases aB dispersing agents, and thus to determine the limitations of this process.
I n a previous investigation (16)of neutral salt solutions as dispersing agents, i t was observed that low concentrations of salts, especially those of calcium and magnesium, had a marked inhibiting action on the dispersion of the protein. Hence the action of dilute neutral salts in combination with acids and bases was studied. It was also considered probable that these data will assist in the interpretation of the dispersing action of salts of strong acids and weak bases, such as aluminum sulfate.
Materials The soybean meal sample was part of the lot used in the neutral salt extraction studies (16) previously reported. Flaked Illini beans were extracted with petroleum ether (boiling a t 30" to 60" C.) in a modified Soxhlet extractor. Both the extraction and solvent removal were carried out a t room temperature. The extracted flakes were ground in a Wiley mill through the 0.5-mm. screen and stored in glass jars also a t room temperature. The ground extracted meal differed from that used previously only in that it had been stored for a longer period of time. The effects of this aging will be discussed later. Brill winter wheat, tepary beans, and Alaska peas were selected for a comparative study. The wheat and tepary bean samples were part of those used in the work previously reported. They had been coarsely ground in a coffee mill, extracted with petroleum ether, and then ground in a pebble mill to pass a 100-mesh screen. The peas were ground directly in a pebble mill t o the same fineness, without previous extraction of oil. The percentage composition of the materials was as follows : Source of Meat5 Soybeans Tepary beans Peas Wheat a
Moisture 9.28 10.31 8.43 11.97
Nitrogen 7.80
4.45 3.80 2.33
Oil 0.35 0.44 0.95 0.57
Ash 5.55 3.18 2.77 1.61
Analytical results are the average of two or more determinations.
The amount of nitrogenous matter extracted from oil-free soybean meal by various acids and sodium and calcium hydroxides was determined over a wide range of pH values. Data are presented to show the influence of hydrogen-ion concentration on the dispersion of the nitrogenous constituents of the meal by sodium chloride and calcium chloride. pH dispersion data for wheat, tepary beans, and Alaska peas are given for comparison.
Protein Extraction
Two and one-half grams of the meal and 100 ml. of the dispersing solution were placed in a 250-ml. centrifuge bottle and shaken mechanically for 30 minutes. The dispersions were centrifuged for 6 minutes in a centrifuge developing a maximum relative centrifugal force at the bottle tip of 1975 times gravity, and a 1 A coijperative organization participated i n by the Bureaus of Chemistry 10-ml. aliquot of the supernatant liquid was removed for analysis. and Soils and of Plant Industry of the United States Department of AgriLonger centrifuging under these conditions did not change the reculture, and the Agricultural Experiment Stations of the North Central sults. All nitrogen determinations were run in duplicate by the States of Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, official A. 0. A. C. Kjeldahl-Gunning-Arnold method with a preNebraska, North Dakota, Ohio, South Dakota, and Wisconsin. 1414
DECEMBER, 1938
INDUSTRIAL AND ENGINEERING CHEMISTRY
1415
TABLEI. TOTALNITROGEN EXTRACTED FROM OIL-FREESOYBEAN MEALBY ACIDS --Hydrochloric-Me." N acid/g. dispersed, meal pH % 0.0 6.6 84.8 6.2 64.7 0.073 5.8 37.1 0.146 12.7 0.293 5.1 0.440 4.6 9.3
--TrichloroaceticMe. acid/g. meal pH 0.0 6.6 0.092 6.0 5.5 0.184 0.368 4.8 0.508 4.5
Sulfuric-
84.8 60.8 22.2 11.0 8.5
pH 6.6 6.6 6.5
6.8 6.0
85.5 84.3 82.8 75.7 65.9
%
dispersed,
%
-PhosphoricMe. acid/g. meal 0.0 0.088 0.175 0.350 0.456
pH 6.6 6.4 6.2 5.8 5.5
N dispersed,
--
Oxali-
84.8 75.0 66.0 48.6 22.9
Me. aoid/g. meal 0.0 0.097 0.194 0.389 0.584
pH 6.6 6.0 5.5 4.9 4.4
%
N %
dispersed, 84.8 61.5 27.6 11.0 7.4
4.2 3.8 3.3 2.9 2.3
8.5 10.5 51.0 72.6 83.3
0.60 0.736 0.920 1.060 1.244
4.2 3.8 3.3 3.0 2.6
7.9 8.1 10.2 24.4 44.1
0.122 0.163 0.245 0.326 0.400
5.8 5.6 5.2 4.9 4.7
53.6 31.7 15.8 13.4 11.8
0.876 1.052 1.316 1.752 2.188
5.0 4.8 4.6 4.1 3.7
12.8 11.6 10.1 9.1 10.7
0.776 0.972 1.216 1.460 1.944
4.1 3.8 3.4 3.2 2.8
6.7 7.4 9.5 15.8 62.9
2.196 2.93 3.66 7.32 10.98 18.30
1.7 1.5 1.3 1.0 0.8 0.5
84.2 84.5 82.7 75.5 60.9 53.7
1.380 1.84 2.30 2.76 3.22 3.68
2.4 1.8 1.6 1.5 1.2 1.2
50.9 46.0 38.7 35.0 27.0 12.6
0.408 0.490 0.571 0.653 0.734 0.816
4.6 4.5 4.2 4.0 3.8 3.6
11.8 10.7 10.1 10.1 11.0 11.5
2.728 3.50 4.38 8.76 13.14 17.52
3.4 2.9 2.7 2.1 1.9 1.7
24.0 72.1 80.0 82.7 83.4 83.6
2.432 2.92 3.89 4.86 9.72 14.58
2.5 2.3 2.0 1.7 1.3 1.2
72.8 77.9 81.1 78.0 77.7 78.7
1.1 0.8 0.7 0.5
9.8 7.0 5.7 5.3 3.8 4.9 93.9
0.898 1.224 1.632 2.448 2.856 4.00
3.4 2.7 2.3 1.9 1.6 1.5
13.1 37.2 62.4 68.4 69.3 70.3
21.90
1.6
80.6
25.10
1.0
67.1
Milliequivalents.
b Data furnished by
... ... ...
R. H. Nagel and H. C. Becker of this laboratory.
NITROGEN EXTRACTED FROM OIL-FREE MEAL TABLE 11. TOTAL BY SODIUM AND CALCIUM HYDROXIDES --NaOH Me." base/ g. meal 0.01'7 0.084 0.168 0.420 0.840 1.68 2.94 4.20
pH 6.8 7.3 8.3 10.5 11.4 12.2
... ... ...
.. ..
.. .. ..
e
N
Me. acid/g. meal 0.0 0.004 0.016 0.040 0.082
0.584 0.732 0.916 1.100 1.464
4.60 9.20 13.80 18.40 40.0b 68.0b 160.0b 4
7 -
N
dispersed,
.... .. ....
N disperaed, 84.6 87.7 91.5 93.9 95.8 96.5 95.2 95.5
.. .. .. .. ..
-
c
Me base/ g. meal 0.0 0,007 0.014 0.028 0.042 0.056 0.070 0.141 0.176 0.352 0.704 1.056 1.76
Ca(0H)z
PH 6.6
6.6 6.6 6.7 6.8 6.9 6.9 7.3 7.7 9.8 10.8 11.2 11.7
N dispersed, % 82.6 81.9 82.5 83.7 83.8 83.7 84.7 86.0 87.0 89.2 92.2 91.5 86.9
Milliequivalent&
cision of 1 per cent or better of the total extracted nitrogen, calculated on the basis that the aliquot contained one-tenth of the total dispersed nitrogen. The temperature of the extracting solutions varied from 24' to 26' C. The nonprotein nitrogen was not determined but was reported for the soybean by Hamilton (6) as 5.55 per cent of the total nitrogen. The hydrogen-ion concentrations were determined with a portable glass-electrode pH meter. All pH values above 9 are recorded as corrected against standard buffers. Anallytical data are reported without correction for moisture, and the nitrogen is given as percentage of total nitrogen in the meal.
Protein Peptization The first series of extractions was made with hydrochloric, trichloroacetic, sulfuric, oxalic, and phosphoric acids, and with sodium and calcium hydroxides a t various concentrations to determine the effect of hydrogen-ion concentration on the amount of total nitrogen dispersed. These data are recorded in Tables I and I1 and plotted in Figure 1. A second series of extractions was made using 0.01, 0.1, and 0.5 N solutions of sodium chloride in which the p H was varied by addition of hydrochloric acid or sodium hydroxide. These data are shown in Table I11 and Figure 2. A third series of extractions was carried out, using 0.001, 0.0175, 0.1, and 0.5 N solutions of calcium chloride in which the p H was varied by the addition of hydrochloric acid or calcium hydroxide. Results of this series are given in Table IV and Figure 3. A fourth series of extractions was made in order to compare the dispersion of soybean protein with that of other vegetable proteins by the same technique. ' I n this series wheat, tepary beans, and Alaska peas were extracted
TABLE111. TOTALNITROGENEXTRACTED WITH SODIUM CHLORIDE SOLUTIONS IN WHICH THE PH Is VARIED BY THE ADDITION OF HYDROCHLORIC ACIDOR SODIUM HYDROXIDE Me." of Acid or Baae/C. of Meal 0 0.080 0.160 0.320 0.560 0.800 1.000 1.200 1.600 2.00 2.40 4.00 8.00 12.00 16.00
0.016 0.032 0.112 0.241 0.802 2.00 3.21 5
-0.01 PH 6.5 5:5 4.9 4.2 3.5 3.1 2.8
i:s i:2 0.9 0.5 0.3
N NaCl-
N extd.,
%
PH
N NaClN extd.,
%
Hydrochloric Acid Added 68.9 6.4 39.9 30.4 6.0 22:4 23.7 5.5 13.5 17.3 5.0 10.7 4.2 14.4 27.2 13.3 3.4 65.3 51.6 3.2 71.4 78.3 2.7 2.1 76.3 82:l i:5 76:3 77:7 1.2 74.9 72.6 0.8 70.4 65.0 0.7 56.7 52.1 0.5 53.9 Sodium Hydroxide Added
6:s
79:2
i:9 11.1
90:3 94.9 95.0 95.2
....
-0.1
6:7 7.3 9.1 11.3
.. ..
43:9 51.7 67.8 91.7 93.3 94.8
-0.5
N NaCIN extd.,
PH
%
6.1 5.6 5.3 4.8 4.1 3.6 3.1 2.7 2.0
73.8 70.4 70.3 57.9 42.3 60.5 67.7 69.6 68.6
i:5 1.1 0.7 0.5 0.4
67:7 67.2 64.9 63.6 61.5
6.2 6.4 7.0 8.7 11.2
72.4 73.3 75.2 80.5 91.4 93.3 93.3
....
Milliequivalents.
with hydrochloric acid and sodium hydroxide solutions of various concentrations. These data are given in Table V.
Effect of Meal Age The well-known instability of protein systems suggested the desirability of determining, after various intervals of time, the percentage of nitrogen and of total water-extractable nitrogen in the ground soybean meal samples. (During the course of this investigation Jones and Gersdorff reported on "Changes That Occur in the Protein of Soybean Meal as a Result of Storage," IO.) The results are given in Table VI as well as data on a few extractions with 0.5 N sodium chloride solutions. These results show a small increase (from 7.80 t o 8.05 per cent) in the nitrogen content of sample A which was ground in June, 1937. This increase may be accounted for by a corresponding loss of moisture. Sample B was flaked and extracted a t the same time as sample A but was not ground until October, 1937. Its nitrogen content remained constant a t 8.14 per cent.
1416
IRTDUSTRIAL AND ENGINEERING CHEMISTRY
VOL. 30, NO. 12
tween October 5, 1937, and January 11, 1938. I n this period the nitrogen extracted by water (single extraction technique) decreased about 3.4 per cent. The time for the collection of data for any single dispersion curve was limited to one week. Recently Jones and Gersdorff (IO) reported that “the amount of nitrogen which can be extracted (from soybean meal) with neutral salt solution decreases with the aging of the meal.” They also reported a decrease in the digestibility of the nitrogen i n vitro. Water was used as the extracting solution for most of the observations on aging recorded in this paper. Consequently the data obtained are not directly comparable with those of Jones and Gersdorff. However, the few salt solution extractions made agree well with their data, despite differences in technique. Jones and Gersdorff also showed the effect of temperature on the stability of the protein. Other factors which may markedly influence this stability are the moisture content of the meal and the influence of light. 3
Effect of Hydrogen-Ion Concentration
EXPERIMENTAL EQUIPMENT FOR PRODUCING OIL-FREE SOYBEAN MEAL
The p H dispersion curves for hydrochloric, sulfuric, oxalic, and phosphoric acids are very similar, showing a minimum dispersion of about 8 per cent of the total nitrogen at a p H of 4.1 to 4.2, and a maximum dispersion of about 83 per cent a t a p H of 1.8. As the p H drops below this value, there is a rapid decrease in the amount of nitrogen dispersed. Trichloroacetic acid is considered a protein precipitant (6) and has been used in the determination of nonprotein nitrogen in the soybean. I n dilute solution, however, it behaves much like the other acids. The dispersion curve for this acid shows approximately the same minimum point at p H 4.2 as the other curves, but after reaching a maximum of 51 per cent a t a p H of 2.3, it drops off rapidly t o 5.5 per cent a t p H 0.5. Therefore trichloroacetic acid should be used in sufficient concentration to produce a p H below 0.5 if it is employed as a precipitant for soybean protein in the determination of nonprotein nitrogen. Table I shows that below this p H the dispersion curve for trichloroacetic acid levels off a t the point a t
Although the effect of age on the nitrogen content of the meal is apparently negligible, this is not true for the waterextractable nitrogen which shows a steady decrease with aging. This decrease amounts to approximately 1.1 per cent per month under the present conditions of storage and for the time interval covered, as determined by the single extraction technique. The decrease in water-extractable nitrogen on aging, as determined by the technique described in the previous paper (15) and consisting of three successive extractions, is much less-namely, about 0.5 per cent per month. The few available data on extractions by 0.5 N sodium chloride indicate, upon aging of the meal, a larger change (decrease) in salt-extractable nitrogen than in waterextractable nitrogen. The previous paper showed that the amount of salt-extractable nitrogen from meal ground in a pebble mill was much less than that obtained from meal ground in the Wiley mill, although the waterextractable nitrogen was about the same for the two meals. It appears that intensive grinding and aging have somewhat similar effects on the quantity of nitrogen that can be extracted from the meal. The data in Table VI indicate that o Hydrochloric Acid, HCl the decrease in extractable nitrogen 0 Sodium Hydroxide, NaOH with aging is greater in the ground 0 Trichloroacetic Acld, HOpCeCIs meal than in the flaked meal. However, the storage conditions of the two €4 C a l c i u m H y d r o x i d e , Ca(0H)e meals were different and may be responsible for this discrepancy. The ground meal was stored in glass bottles exposed t o diffuse light, whereas the flaked meal was stored in cans proteited from light. pH of E X T R A C T The experimental work included in this paper, with the exception of the TOTAL NITROGEN EXTRACTED FROM OIL-FREESOYBEAN MEALBY ACIDS FIGURE 1. AND BASES sulfuric acid data, was performed be-
DECEMBER, 1938
INDUSTRIAL AND ENGINEERING CHEMISTRY
1417
which approximately 5 per cent nitrogen is exTABLEIV. TOTAL NITROGEN EXTRACTED WITH CALCIUM CHLORIDE SOLUtracted, and only in very concentrated acid does TIONS IN WHICHTHE PH Is VARIED BY THE ADDITIONOF HYDROCHLORIC it rise again. ACID OR CALCIUM HYDROXIDE The most efficient dispersion appears to be Me.Q of -0.001 N CaCI2- -0.0175 N CaCh- -0.100 N CaC12- -0.500 N CaClzproduced by sodium hydroxide. The dispersion Acid or N N li N extd., extd., extd., extd., Base/G. curve for this base is slightly above that for calof Meal pH PH % PH % PH % % cium hydroxide which a t higher concentrations Hydrochloric Acid Added points downward quite sharply; this behavior 5.3 24.9 5.2 66.3 0 6.6 75.5 5.7 17.9 5.4 15.8 5.0 25.3 4.8 67.9 0.0800 6.0 56.0 indicates that larger quantities of lime might 27.4 5.2 14.9 4.7 27.1 4.6 67.5 0.1598 5.6 11.9 4.4 31.2 4.2 69.2 0.3197 4.9 markedly decrease the amount of nitrogen dis0.4795 413 13:3 4.1 47.1 3.9 71.1 persed. 0.5594 4:1 s:5 .. .. , . .. The acid and base form one continuous curve 65.6 3.6 72.5 0.6394 3.9 12.7 3.8 3.3 0.7992 3:4 12:s 3.6 21.3 3.5 73.8 72.7 on which the amount of nitrogenous matter dis61.0 3.2 59.1 3.1 77.5 2.9 73.0 0.9920 3.0 persed by water is a single point. The maximum 74.4 2.9 74.0 2.5 78.1 2.6 69.6 1,199 2.6 2.1 2 . 0 1.598 2.3 80.5 79.6 69.7 amount of nitrogen dispersed by acid is slightly 1.998 i:s 80:7 2.0 80.8 1.8 1.8 79.8 67.3 less than that produced by water, which dis1.7 82.0 1.6 80.4 1.6 65.5 2.398 . . .. perses about 84 per cent. 1.4 .. 1.4 79.0 1.3 64.6 3.197 1.1 65.1 3.996 i:i 77:2 1.2 .. 1.2 76.6 The p H at the minimum point of solubility 72.3 59.6 71.3 0.8 0.7 0.7 7.990 0.7 0.5 6i:4 11.99 0.5 63.1 (approximately p H 4.2) is considered to be an 0.5 58.8 0:3 5s:1 0:4 5810 15.98 0.3 53.1 isoelectric point for the protein in soybean meal Calcium Hydroxide Added under the conditions of these experiments. 5.7 17.6 0.014 5:3 25:s 76:2 5:4 6718 0,018 6: 6 Purified soybean protein was reported to have 5.5 25.5 5.4 66.3 78.3 0.035 6.7 an isoelectric point of p H 5.02 by Hartman and 25.7 5.5 .. .. 5.6 67.5 79.8 0.053 6.8 25.7 67.1 5.7 .. .. 5.7 80.7 0,070 6.9 Cheng (7) and p H 4.7 by Csonka, Murphy, and 6.0 26.4 6.1 64.3 81.9 0.106 7.1 Jones (4). The minimum point on the curves 26.3 6.3 65.9 83,3 6:s 2o:o 6.7 0.141 7.4 here corresponds closely to the isoelectric point .. 6.8 64.4 7.3 21.3 .. 0.176 . . . .. . . .. 7.8 22.1 0.211 of approximately p H 4.1 found for commercial s:3 2s:1 .. .. 84:7 0,246 s:9 soybean flours by a cataphoretic method, as re9.3 30.3 8.9 65.6 9.3 28.2 86.5 0,382 9.8 ported by Monaghan-Watts (12). Hence the 10.1 71.1 10.4 57.8 10.6 78.5 89.9 0,704 10.8 11.1 90.4 11.0 62.0 10.8 69.4 89.0 1.06 11.3 solubility data furnish additional evidence that 11.4 63.2 11.2 68.0 11.5 81.9 1.41 11.6 87.7 the isoelectric point of crude protein in soybean a Milliequivalents. meal occurs a t a lower p H value than that of the refined protein. This difference may be accounted for bv the txesence of substances as(11) claimed that a t any given p H value the reaction between sociated with the protein in the meal or possibly by changes salts and proteins was stoichiometric and was governed only in the protein during the process of separation from the meal. by the valence of the ion with charge opposite to that of the Combined Effect of pH and Salts protein. He believed that the earlier data claiming a lyotropic series was in error because of lack of control of the hyThe influence of p H on the salt dispersion of proteins has drogen-ion concentration. Gortner 16) and others have taken been given considerable attention in the literature. Loeb a different point of viek, and have produced considerable evidence to support their contention of a lyotropic series effect, especially in relation to the dispersion of proteins by salts. I n extracting protein from wheat flour with 0.5 N magnesium sulfate, Rich (IS) showed that the effect of adding small amounts of acid to the salt was practically negligible. The present data are intended as a step toward the interpretation of the relative effects of the hydrogen ion and salt ions on protein dispersion. The first paper of this series (16) presented data on the effect of neutral salts without added acid or base. That some reaction occurs between the meal and the salt was indicated by a decrease in the p H value of the extract with an increase in salt concentration, even though no acid was added. This change in pH may be caused by a reaction between the negative protein particles and the cation of the salt, resulting in an increase in the hydrogen-ion concentration. The fact that divalent cations had more effect than monovalent cations would seem to lend support to this hypothesis. With respect to the superimposed effect of FIGURE 2. TOTAL NITROGEN EXTRACTED BY SODIUM CHLORIDE SOLUTIONS p H on the salt dispersion of the protein, it is clearly evident from these data that a t low salt IN WHICHTHE PH Is VARIED BY ADDITION OF HYDROCHLORIC ACID OR SODIUM HYDROXIDE concentrations the effect of hydrogen-ion con-
INDUSTRIAL AND ENGINEERING CHEMISTRY
1418
- o mO.OO1
N CoClz and HCI or Co(OHlz
m0.0175N CoClzond HCI or Co(OH),
A A 0.1 0 0
0
I
2
3
5
4
6
7
N CoCleond HCI or Co(OH),
ride concentrations, however, the effect of pH would practically disappear. The charge on the protein micelle is intimately related t o its stability in solution, as shown by loss in dispersibility a t the isoelectric point determined by acid precipitation. This minimum point broadens out as the salt concentration is increased and practically disappears for 0.5 N calcium chloride. The question arises as to whether a change in pH is any longer effective in neutralizing the micellar charge in the more concentrated salt solutions, and whether in such solutions the micellar hydration is increased. The answer, at least in part, possibly lies in studies of these systems by the method of electrophoresis. The data on nitrogen extraction for the Alaska pea and tepary bean (Table V) are similar to those for the soybean meal; those for the wheat show more resistance to the dispersing action of the acid and alkali.
Summary
0.5 II CoC1,ond HCI orCafO% 8
9
IO
VOL. 30, NO. 12
II
The amount of nitrogenous matter extracted from oil-free soybean meal by hydrochloric, triFIGURE 3. TOTAL NITROGEN EXTRACTED B Y CALCIUM CHLORIDE SOLUTIONS chloroacetic, oxalic, sulfuric, and phosphoric IN WHICHTHE PH Is VARIEDBY ADDITION OF HYDROCHLORIC ACIDOR acids, and by sodium and calcium hydroxides CALCIUM HYDROXIDE was determined over a wide range of pH values. These data show that the maximum centration is the greater; a t higher salt concentrations (0.5 N amount of protein extractable by the acids studied is no for calcium chloride) the effect of pH practically disappears. greater than the amount extractable by water (approxiA concentration of 0.5 N for sodium chloride does not elimimately 84 per cent at 26’ C.), and that alkali is the most nate the pH effect as completely as does 0.5 N calcium chloride, effective dispersing agent, extracting up to 95 per cent of the but it is very small between pH 5.2 and 8.0. The most pronitrogenous matter at even moderate concentrations. The acids have a common point of minimum dispersion a t pH nounced effect of pH is between 3.0 and 5.2 and above 8.0. 4.2, approximately. The trend of the data indicates that a t higher sodium chloExtractions made with salt solutions in which the pH was varied by addition of hydrochloric acid in conjunction with sodium chloride or calcium chloride, show that a t low salt T A B LV.~ TOTAL NITROGEN EXTRACTED FROM ALASKAPEA, concentrations the pH has a greater influence on the amount TEPARY BEAN, AND WHEATMEALSBY HYDROCHLORIC ACID of protein dispersed than the salt. As the salt concentration AND SODIUM HYDROXIDE is increased, the effect of the acid decreases and practically Me.” Acid -Alaska Peas-Tepary Beans-Wheatdisappears a t 0.5 N for calcium chloride. or Base/G. N extd., N extd., N extd., Meal PH % PH % PH % Aging of the meal has been found to decrease the quantity Hydrochloric Acid Extraction of water-extractable nitrogen. This decrease per month 1.181 2.0 82.8 2.0 86.3 1.7 50.9 amounts t o about 1.1per cent of the total nitrogen. Hydro0.551 2.8 51.9 3.1 69.9 2.1 47.1 0.315 3.9 14.8 4.0 22.7 2.5 42.8 gen-ion dispersion data for wheat, tepary beans, and Alaska 0.1576 4.9 19.3 4.9 24.7 3.5 23.1 0.0000 6.7 77.9 6.5 78.2 6.4 20.2 peas are given for comparative purposes. pH of E X T R A C T
Sodium Hydroxide Extraction 0.0802 0.1603 0.401 a
8.0 9.1 10.5
7.6 8.7 10.4
94.7 97.6 97.7
93.2 94.9 99.3
9.1 10.1 11.0
54.2 89.0 97.3
Milliequivalents.
TABLE VI. 0
Sample A=
EFFECTOF AGINGOF MEALON THE QUANTITYOF EXTRACTABLE NITROGEN Date of Extn.
% ’ N at Date of Extn.
r
in Meal
6- 7-37 7.80 7-29-37 7.80 12- 4-37 7.80 2-21-38 8.05 BO 10- 7-37 8.14 11-19-37 8.14 12- 4-37 8.14 1-10-38 8.14 Cb 2-21-38 7.31 a Samples A and B were taken from
’% N Extd. by: -NaClThree One 100-ml. 100-ml. 100-ml. 100-ml. extns. extn. extns. extn.
F-HIO--
Three 90.1
..
One
si:,
84.1
..
.. ..
81.1 86:2 79.7 7i:6 69:i 90.0 84.8 82.6 81.8 7i:4 80.7 .. 89.9 85.1 84.3 80.8 the same lot of beans of the 1936 crop. b Sample C was the same variety as A and €3 but came from the 1937 crop.
.. .. ..
.. .. ..
..
..
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