1QS8
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
Vol. 41, No. 5
of the difficulty of maintaining steady conditions for a period sufficient t u give reliable and reproducible results. The maximum rate of boilup that could be obtained from a 5-liter flask heated with a Glas-Col heater was 7500 1111 per hour. This was not, sufficient t o flood Column 3 (Table I and Figure 4 ) ; hence the characteristics of this column are shown in Figure 4 only up to this boilup rate and not t o its flood point,. The behavior of Column 3 a t the boilup rate of 7500 nil. per hour indicated that its maximum capacity was a t least 8 or 9 liters per hour.
Any attempt to extrapolate the operating characteristics as shown in Figures 2 to 4 to colunins of other diameters is complicated by several factors: The number of disks per foot of column was not constant; the percentage of the area of the disks cut out t o form the vapor path was not exactly const,ant; and the ratio of the diameter of t,he disks t o t h a t of the tubing \?-as not coilstarit in the three columns described. However, a few qualitative statements about the effect of increasing the diameter on operating characteristics seem justified. The number of theoretical plates per foot is markedly reduced a t larger diameters, bot,h because of fewer disks per foot being used and because of increasing difficulty of maintaining close contact between disks and tubing. Pressure drop per foot of column decreases, primarily because of the presence of fewer disks per foot. The liquid holdup increases somewhat. The greater area of wet surfaces accounts for this, but the effect is diniinished bj- lhe presence of fewer disks per foot.
L a
w
W v)
4a -I
2 W B:
8 I-
Like other types of screen-packed columns, it is essential that the packing bc thoroughly and uniformly wet before beginning a distillation or test run. This is accomplished easily by a short period of rapid boilup under total reflux. T h e n wet, a uniform continuous film of liquid covers all screen surfaces. This film remains intact so long as reflux is continucd, no mat,tcr how low the boilup rate. Maximum plate efficiency was obtained a t very low boilup rat'es (barcly cnough to keep the pa,clting wet,). LITERATURE CITED
netted to the still pot and open to the atmosphere on t,he other arm. Holdup was measured by the niethod recommended by Ward; heptane and biphenyl ether were used and analyses were made by refmct.ive index. The rate of boilup and t,he capacity of the columns were determined by operating with total take-off for a brief measured interval (0.5 or 1.0 minute) and measuring the volume of distillate. Thus, the rates were measured a t the still head and not at, the pot, though there should be little difference in rates a t the two points. S o tests were run beyond t,he point of incipient flooding because
(1) Bragg, L. B . , I K D . E X G . CHEM., ~ " I L . ED.,11, 283 (1939). (2) Grisn-old, John, Morris, J. K., and Van Berg, C. F.,IXD.ENQ. CHEY.,36, 1119 (1944). (3) Lecky, H. S., and Ewcll, R. H., ISD.ENC.CHIIM.,AXAI..ED.,12, 644 (1940). (4) Stedman, D. F., X a t l . Petroleiim S e w s . 29, 11-125-6. 11-128 (Aug. 25, 1937). ( 5 ) Ward, C. C., U . S.Bur. Mines, Tech. Paper, 600 (1939). ( 6 ) Willingham, C . B.. and Rossini. E. D., J . Reseaich .Vall. Bur. Szandards, 37, 15 (1916). R E C E I V E .4pril D 3, 1948. The nienrion of comiiierciril products does not iiiiplp t h a t they are endorsed 01' recommended by the Dcpartment of Agriculture over others of a similar nature not mentioned.
L. L. AICKIXNEY AND W. F. SOLLARS' .Vorthern Regional Research Laboratory, Peoria, I l l .
I
N COKXECTIOX with studies of the nonenzymatic browning
on soybean protein, it was desired to inveatigate the extraction of protein from solvent-extracted soybean meal with sulfurous acid. Although i t was known that sulfurous acid could be used t o peptize the protein (6, Q), quantitative information had not been reported. Also, it had been claimed ( 6 ) t h a t soybean protein, extracted with sulfurous acid, exhibited a superior light color. Soybeans contain about 9% sugars ( l a ) . Only a small amount 1
Present address, Purdue University, Lafayette, Ind.
of reducing sugars are present (0.1 to 0.2%) ( 7 ) . It is possible, therefore, that part of the bromn color observed in soybean protein obtained from hexane-extracted meal Tvithout the use of sulfur dioxide is caused by the well-known "browning reaction." In order t o prevent the formation of this color, sulfur dioxide is used t o precipitate the protein from the meal extract ( I O , I S ) . Sodium sulfite is often used along with alkali in extracting the protein from the meal (6,8 ) . Another method involves removing sugars by leaching the solvent-extracted meal with aqueous sulfurous acid a t the isoelectric point of the protein (9, 13).
INDUSTRIAL AND ENGINEERING CHEMISTRY
May 1949
% ,
T h e extraction of protein from soybean meal with sulfurous acid is reported. Peptization curves show that more than 8Oy0 of the total nitrogen of hexane-extracted meal is peptized between pH 1.5 and 3.0. The protein of ethyl alcohol denatured meal is not so readily peptized with sulfurous acid. Protein extracted with sulfurous acid does not exhibit the brown color which is associated with nonenzymatic browning. A mixture of hydrochloric and sulfurous acids may be employed to extract protein and still inhibit the browning reaction, thus eliminating the need of a closed system to prevent the escape of sulfur dioxide when used in the required concentrations.
hIATERIALS AND METHODS
SOYBEAN MEAL. For laboratory studies, Illini beans, 1943 crop, were cracked, dehulled, flaked to a thickness of 0.005 to 0.007 inch, and air-dried to 10% moisture in preparation for oil extraction. Oil was removed by a Soshlet extractor with hexane or absolute ethyl alcohol. The extraction time was 8 hours, wit8h the temperature of the flakes during extraction held a t approximately 30" C. (86" F.) for hexane and 65" C. (149" F.) for absolute alcohol. The flakes were stripped of solvent by air drying. For pilot plant studies, a commercial petroleum ether-extracted flake of high nitrogen solubility was used. NITROGEN.The method of Smith and Circle (11)was used for determining the percentage of total meal nitrogen peptized at various p H value~-2.5-gram portions of ground oil-free meal contained in 250-mi. centrifuge bottles, were shaken for 2 hours a t room temperature with 100 ml of water containing sufficient sulfur dioxide to give the desired p H value, and then cmtrifuged. An aliquot of the clear, supernatant liquor was taken for nitrogen analysis, and nitrogen determinations were made by the Association of Official Agricultural Chemists' Kjeldahl-Gunning-Arnold method, with a rnerrury catalyst and 1-hour digestion. The p H values were determined by means of a glass electrode. ISOLATION OF PROTEIN. For the pilot plant preparation (experiment 4), 100 pounds of flakes were slurried with 2000 pounds (240 gallons) of tap water and sulfur dioxide was added t o reduce the p H to 2.2. Twelve pounds of sulfur dioxide were required. After stirring for 1 hour, the slurry was run over a n 80-mesh, 2 X 4 feet, gyrating screen tilted a t an angle of 6" to remove thegreater portion of insolubles. The extract was then clarified at 7600 r.p.m. by a disk centrifuge of the valve-bowl concentrator type with automatic controls for solids discharge. The sludge was continually recirculated to reduce dispersion loss. T h e protein was precipitated by adjusting the p H of the clarified extract to 4.2 with 20% sodium hydroxide solution. After settling over night, t h e concentrated protein curd was filtered on a vacuum drum filter equipped with a string discharge. The wet protein curd cono tained 64y0 moisture and was dried in a forced draft oven at 120
.- ,Hexane
Extracted Flakes
x I40
I / 0
P
..
5 rot0
Abr. E t O H Extracled F l a k e s A \
I
3
4
5
pH OF EXTRACT
Figure 1. Peptization Curves Showing Percentage of Total Nitrogen Extracted from (a) Hexane and ( x ) Absolute Ethyl Alcohol-Extracted Soybean Nleal
1059
t o 130' F. Details of the pilot plant operation are reported elsewhere (9). With the exce tion of experiment 7, the laboratory reparations listed in Table ?were extracted by slurrying the oil-gee meal (or leached meal) with 20 parts by weight of di~tilledwater and then adjusting the p H to 2.3 * 0.1 with sulfur dioxide. I n experiment 7, the same extraction procedure was used except that sulfur dioxide was added t o pH 3.2 and then dilute hydrochloric acid was added to p H 2 2. The insoluble residue, after stirring for 1 hour, was removed by centrifuging for 6 minutes a t 2000 times the normal force of gravity. Where three successive extractions were made (experiments 1, 5, and 6), the residue was slurried with t h e same quantity of distilled water as used in the first extraction, and the p H was adjusted t o 2.3 * 0.1 with sulfur dioxide. After a n hour's stirring the residue was again removed by centrifuging. The third e x t r a d o n was then carried out in the same manner as the second. I n experiments 3 and 6, the acid extract was dialyzed against distilled water until free of sulfite (7 days) and the protein recovered from the dialyzer contents by centrifugation. I n experiments 1, 2, 5, and 7, the protein was recovered from the extract, or combined extracts in case of three successive extractions, by adjusting the p H t o the isoelectric region with 10% sodium hydroxide solution and centrifugation a t 2000 times the normal force of gravity.
TABLE I. ANALYSIS O F PROTEINS EXTRACTED WITH SULFUR DIOXIDE
Experiment No.
1
Oil Extraction Hexane
2
Hexane
3
Hexane
Protein YieldQ, Ash, Pb, Nb, Preparation % N % % % 3 extractions; pptd. at pH 4.2 85 0.28 0.80 15.25 Leached with SO2 a t pH 4.2; 1 extraction; pptd. at p H 4.4 55 0.74 0.85 15.90 Leached with SO2 at D H 4.2: 1 ex-
Hexane Abs. CzHaOH Abs. CzHsOH Hexane
42
0.21
25
1.18 0.51 15.45
60 lysed 1 extraction b y adding 902 to pH 3.2 then HCI to p H 2.2; pptd. a t pH 4.2
...
0.82 15.82 0.76
...
...
0 . 2 3 0.65 16.12
50
0.42
...
15.97
Percentage total nitrogen reoovered as protein. b Corrected for ash a n d moisture.
0
PEPTIZATION WITH SULFUR DIOXIDE
T h e percentage of total nitrogen peptized by various concentrations of aqueous sulfur dioxide from solvent-extracted soybean meal is plotted in Figure 1. The lower curve is t h a t of absolute ethyl alcohol-extracted meal while the upper curve was obtained from hexane-extracted meal. The lower percentage of nitrogen peptized from the alcohol-extracted meal is characteristic of the denaturing effect of alcohol on protein. The extent of denaturation of the protein and hence the shape of the dispersion curve for ethyl alcohol-extracted flakes varied with time and temperature of the ethyl alcohol extraction The moisture content of the flakes also was found t o be a factor in denaturation: less denaturation was observed with flakes containing 3 t o 4Cr, moisture than when 10% moisture was present. With hexane-extracted meal, a maximum of 87y0 of the'total nitrogen was extracted at p H 2.5. The maximum is rather broad, so t h a t more than 230% of the nitrogen is extracted at any p H between 1.5 and 3.0. Thirteen and one-half milliequivalents of sulfurous acid were required t,o effect a p H of 2.5. The peptization curve for alcoholextracted flakes indicates t h a t sulfur dioxide is not a practical extracting agent, for a meal that has been treated in such a manner as t o denature the protein. The peptization curve for sulfurous acid is similar t o that reported for hydrochloric acid and the maximum peptization is much greater than for sulfuric acid ( 6 , l i ) .
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INDUSTRIAL AND ENGINEERING CHEMISTRY
ISOLATION OF PROTEIN BY SULFUR DIOXIDE EXTRACTION
The analyses of proteins obtained by varying the procedure of isolation are listed in Table I. T h e yield is reported as per cent of total nitrogen of the meal recovered in the isolated protein. Where three successive extractions were made (experiments 1, 5 , and 6) the yields obtained compared favorably with those expected from the peptization curves. The phosphorus content is high, indicating t h a t the protein is contaminated with 1.7 t o 3.0% phytic acid. The presence of phvtic acid was expected (6). In experiments 2 and 3, the flakes were leached at the isoelectric point of the protein n-ith sulfurous acid solution prior t o extracting the protein. Four successive leachings were made with 20 parts of solvent, resulting in a loss of 33.2% of the solids, including 9.5% of the total nitrogen and 28.9% of the phosphorus. Removal of sugars and other solubles prior t o extracting the protein with sulfurous acid did not produce a protein with greatly improved color when compared t o protein prepared by extracting the meal Tvithout leaching (experiment 1). Experiment 7 was conducted t o determine the feasibility of substituting hydrochloric acid for part of the sulfur dioxide in order t o prevent loss of this volatile agent during extractions. The loss of sulfur dioxide during the pilot plant preparation resulted in a rise in p H from 2.2 t o 2.8 during the operation which required about 6 hours. T h e results of extracting with a mixture of hydrochloric acid and sulfurous acid indicated t h a t identical proteins and yields are obtained; this eliminates the need of a closed system which would be required if sulfurous acid alone were used. The analyses of the protein prepared in the pilot plant are shown under experiment 4 in Tablc I. The only difficulty experienced in the pilot plant operation, other than loss of sulfur d i o d e , was in the clarification of the meal extract. The centrifuge was designed for use with an “oilylilre” alkaline extract. Tlie acid extract did not cxhibit this oily property and there 7%as a tendency for the solids t o build up in the bowl and not discharge properly. COLOR OF SULFUROUS-ACTD EXTRACTED PROTEIN
The effectiveness of sulfur dioxide in inhibiting the browning of soybean protein is demonstrated by adding a small amount of 50% glucose solution t o a small quantity of light colored soybean protein. The mixture is divided and one sample reserved as a bIank, sulfur dioxide or sodium dithionite is added to the other. T h e blank will turn a dark brown in the course of a few days, while the inhibited sample will darken at a much lower rate. Actually, the color of the proteins obtained by sulfur dioxide extraction was disappointing. Although the color was slightly lighter than t h a t of protcins obtained by alkali-sodium sulfitelime extraction and sulfur dioxide precipitation, the difference was only a matter of shade and not outstanding. Proteins extracted with alkali, without the use of sulfite and precipitated with acids other than sulfurous, exhibit a brown color much more pronounced than t h a t of protein extracted with sulfurous acid. This difference in color is in keeping with the inhibition of nonenzymatic browning by sulfites. Proteins from alcohol-extracted meal were light in color. I n fact, the protein obtained in experiment 6 was almost whitc. However, protein prepared by alkali extraction from such meal also contains less color ( 1 , 2 ) . Alcohol-extracted flakes may be compared with hexaneextracted flakes that have been leached a t pH 4.2 with sulfurous acid, in t h a t sugars have been removed in each case. However, protein extracted from alcohol-extracted flakes with sulfur dioxide consistently exhibited a lighter color. The lighter color observed is the result of removing colored constituents, probably pigments and/or phosphatides, during the alcohol extraction because the possibility of nonenzymatic browning has been excluded in each case. The presence of phosphatides was indicated by the fact
Vol. 41, No. 5
t h a t approximately 5% of the phosphorus contained in the protein from experiment 4 was removed by exhaustive extraction with 80 t o 20 ratio of benzene t o ethyl alcohol in a Butts extractor. Protein peptized from petroleum ether-extracted flakes with sulfur dioxide will exhibit a light tan color which is attributable t o the presence of phosphatides and/or natural plant pigments as impurities. The deeper brown color observed in protein which has been isolated without the use of sulfurous acid or sulfites may result from the reaction of reducing sugars with proteinaceous materials. SUMMARY
Peptization curves are presented, showing the percentage solubility of the total nitrogen of hexane- and ethyl alcoholextracted soybean meal in various concentrations of sulfurous acid solutions. Eighty-seven per cent of the total nitrogen in hexane-extracted flakes was peptized at pH 2.5. The denaturing effect of alcohol on protein was exhibited by the fact t h a t only 57% of the total nitrogen in ethyl alcohLL-extracted flakes was peptized (pH 2.2). The peptization curve for sulfurous acid is similar t o that reported for hydrochloric acid. A mixture of sulfurous acid and hydrochloric acid appeared t o be as effective for extracting soybean protein as sulfurous acid alone and more practicahle because of reduced volatility. The proteins extracted with sulfurous acid and Precipitated by adjusting the p H t o the isoelectric region with sodium hydroxide Contained phytic acid as an impurity to the extent of 1.7 to 3.0%. Dialyzing the acid extract failed t o remove appreciable amounts of phytin phosphorus. The color observed in isolated soybean protein appeared to result from natural pigments, phosphatides, and nonenzymatic browning. The brown color was eliminated by extracting with sulfurous acid, and the resulting protein exhibited a light tan color. The light tan color of sulfurous acid-extracted protein was only slightly lighter than t h a t obtained by alkali-sodium sulfite-lime extraction and sulfur dioxide precipitation. Removal of sugar and water solubles from the meal by leaching v-ith sulfurous acid at the isoelectric point of the protein, followed by sulfurous acid extraction, produced a protein of slightly lighter color. However, such protein obtained from hexaneextracted flakes was a deeper color than that obtainrd from u11leached alcohol-extracted flakes. LITERATURE CITED
(I) Beckel, A. C., and Smith A. K., P’oodInds., 16, 616 (1944). K., unpublished experiments. (2) Beckel, A. C., and Smith, (3) Belter, P. A,, Beckel, A. C., and Smith, A, K., IKD.ENG.CHEM., 36, 799 (1944). (4) Cone, C. N., and Brown, E. D., U. S. Patent 1,955,373 (1934). (5) Fontaine, T. D., Pons, 11‘.A , Jr., and Irving, G. TV., Jr., J . RioE. Chern., 164, 487 (1946). (6) Iwamae, H., C . S. Patents 2,272,562 and 2,272,563 (1942). L\
(7) MacMasters, hI. M., Woodruff, S.,and Klaas, H., IND. Eso. CHEM.,13, 471 (1941). ( 8 ) Rauer, P., and Torrington, P., U . S.Patent 3,132,431 (1938). (9) Rawlins, F. G., and Welton, W. M.,Ibid., 2,260,640 (1941). (10) Satow, S., Ibid., 1,321,480 (1919). (11) Smith, A. K., and Circle, S.J., T i m . E K G . CHEX,30, 1414 (1938). (12) Wolfe, A. C., Park, J. B., and Thrrell, R. C., PZant P h y s i d . , 17, 289 (1942). (13) Youtz, 31.h.,U.
S.Patent 2,397,307 (1946).
RECEIVXD March 2.5, 1948.
